Minimally toxic prodrugs

ABSTRACT

The present invention relates to the field of oligopeptide prodrugs that are intended for the treatment of cancer. The selectivity of these prodrugs requires the presence of an (oligo)peptidic moiety and/or a protective capping group to ensure the prod-rug stability in blood. It further in particular relates to the exemplary oligopeptidic moiety ALGP and to prodrugs comprising it. In particular it also relates to the capping group phosphonoacetyl and to prodrugs comprising this capping group.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/133,684, filed Sep. 17, 2018, allowed, which is a continuation U.S.patent application Ser. No. 14/758,002, filed Jun. 26, 2015, now U.S.Pat. No. 10,076,576, issued Jun. 26, 2015, which is the U.S. NationalPhase of International Application No. PCT/EP2013/078034 filed Dec. 27,2013 which designated the U.S. and claims priority to U.S. ProvisionalApplication No. 61/746,621 filed Dec. 28, 2012, the entire contents ofeach of which are hereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:5248.0024_Sequence_Listing.txt; Size: 1.84_kilobytes; and Date ofCreation: Jan. 22, 2020) filed with the application is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of oligopeptide prodrugs thatare intended for the treatment of cancer. The selectivity of theseprodrugs requires the presence of an (oligo)peptidic moiety and/or aprotective capping group to ensure the prodrug stability in blood. Itfurther in particular relates to the exemplary oligopeptidic moiety ALGPand to prodrugs comprising it. In particular it also relates to thecapping group phosphonoacetyl and to prodrugs comprising this cappinggroup.

BACKGROUND OF THE INVENTION

Therapy of cancer remains one of the major challenges of medicine today.Only a combined therapeutic approach will allow this problem to bemastered. This will involve surgery, classical chemotoxic chemotherapy,molecular targeted drugs and immunotherapy.

The major problem in the use of chemotoxic drugs is their lowselectivity for cancer cells resulting in dose limiting and lifethreatening toxic side effects. The most common acute toxicity ismyelotoxicity resulting in a severe leukopenia and thrombocytopenia.Some of the commonly used drugs have also a more specific toxicity.Doxorubicin (Dox), an anthracycline drug, is an 17 of such a chemotoxicdrug that induces besides severe myelotoxicity a severe cardiotoxicity.These toxicities restrict its use above a cumulative dose of 500 mg/m².

Approaches used to increase tumor specificity of a drug are conjugationwith (i) a tumor-recognizing molecule (e.g. receptor ligand; see, e.g.,Safavy et al. 1999— J Med Chem 42, 4919-4924) or with (ii) a peptidethat is cleaved preferentially in the immediate vicinity of tumor cellsby proteases preferentially secreted or produced by tumor cells.

Tumor specific oligopeptidic prodrugs, such as prodrugs of doxorubicin,have been developed.

In contrast to previous studies, these peptidic prodrugs were designedto be impermeable to cell membranes, to remain stable in the blood whilebeing cleaved into the active drug by peptidases released in theextracellular space of solid tumors. These activating peptidases are notnecessarily tumor specific but can increase the drug selectivity to theextent that these peptides are oversecreted in the extracellular spaceof solid tumors and play an important role in cancer cell invasion andmetastasis. The originality of this approach is that it didn't target asingle well known enzyme but all enzymatic activity that was foundexcreted by human tumoral cells maintained in culture.N-succinyl-beta-alanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin(Suc-βALAL-dox) was selected as such a candidate prodrug (Fernandez etal. 2001, J Med Chem 44:3750-3). Compared with unconjugated doxorubicinthis prodrug is, in mice, about 5 times, and in dogs, 3 times lesstoxic. Chronic treatment with Suc-βALAL-dox proved to be significantlyless cardiotoxic than with Dox at doses up to 8-fold higher in rats. Theimproved activity of Suc-βALAL-dox over Dox was observed in severaltumor xenograft models (Dubois et al. 2002, Cancer Res 62:2327-31; Ravelet al. 2008, Clin Cancer Res 14:1258-65). Two enzymes, CD10 (neprilysinor calla antigen) and thimet oligopeptidase (TOP) have been identifiedlater in tumor cell conditioned medium and in tumor cells as activatorsof Suc-βALAL-dox (Pan et al. 2003, Cancer Res 63:5526-31; Dubois et al.2006, Eur J Cancer 42:3049-56) but other non-identified proteases mayalso be involved in the activation process.

A phase I clinical study with Suc-βALAL-dox was initiated by thebiopharmaceutical company DIATOS SA. A myelotoxicity occurred at threetimes higher doses compared with Dox. No drug-related, severe cardiacadverse events were reported, even at very high cumulative doses (2750mg/m²). A clinical benefit was observed for 59% of evaluable patients(Delord et al., unpublished).

The main limitation of Suc-βALAL-dox is that leukopenia remains as animportant toxicity and that experimentally higher antitumoral activitycould only be observed at the cost of a still important myelotoxicity.Such myelotoxicity is expected to occur as the result from thesensitivity of the peptidic moiety of Suc-βALAL-dox to the hydrolysis byenzymes present in normal tissues.

WO 02/100353 specifically discloses chemotherapeutic prodrugs designedwith a 3- to 6-amino acid oligopeptide cleavable by CD10. WO 02/00263discloses prodrugs with a 3-amino acid oligopeptide cleavable by TOP andat least 1 prodrug with an amino acid oligopeptide (Leu-Ala-Gly) notcleavable by CD10. WO 00/33888 and WO 01/95945 disclose prodrugs with a4- to 20-amino acid oligopeptide comprising a non-genetically encodedamino acid at a fixed position, with said oligopeptide being cleavableby TOP. In WO 01/95945, at least 1 prodrug, with a βAla-Leu-Tyr-Leuoligopeptide, was reported to be resistant to CD10 proteolytic action.WO 01/95943 discloses prodrugs with a 3- to 4-amino acid oligopeptidecomprising a fixed isoleucine, said oligopeptide preferably beingresistant to TOP; no information on CD10-susceptibility or -resistanceis given. A more general concept of a prodrug consisting of a druglinked to an oligopeptide (of at least 2 amino acids) itself linked to aterminal group is disclosed in WO 96/05863 and was later extended in WO01/91798.

Other polymeric drug-conjugates of which the non-drug moiety is at leastcomprising a water-soluble polymer and a peptide (comprising 4 to 5natural or non-natural amino acids) selectively cleavable by action ofmatrix metalloproteinases (MMPs) are disclosed in WO 02/07770. WO03/094972 focuses on anti-tumor prodrugs that are activatable by thehuman fibroblast activation protein (FAPα); the prodrug comprises anoligopeptide of 4 to 9 amino acids with a cyclic amino acid at a fixedposition. WO 99/28345 discloses prodrugs that are proteolyticallycleavable by prostate-specific antigen in the oligopeptide of less than10 amino acids present in the prodrug.

WO 97/34927 revealed the FAPα-scissable prodrugsAla-Pro-7-amino-4-trifluoromethylcoumarin andLys-Pro-7-amino-4-trifluoromethylcoumarin. WO 00/71571 focuses onFAPα-scissable prodrugs, with some further experimental investigationson proteolytic sensitivity to CD26 (dipeptidylpeptidase IV), the latterbeing considered as undesirable due to the relative abundance of CD26also in non-malignant cells.

Other prodrugs activatable by FAPα include promellitin toxin (LeBeau etal. 2009, Mol Cancer Ther 8, 1378-1386), doxorubicin (Huang et al. 2011,J Drug Target 19, 487-496), thapsigargin (Brennen et al. 2012, J NatlCancer Inst 104, 1320-1334), and a prodrugs comprising an oligopeptideof 4 to 9 amino acids with a cyclic amino acid at a fixed position (WO03/094972).

WO 01/68145 discloses MMP-cleavable but neprilysin (CD10)-resistantdoxorubicin prodrugs (see Example 1001 therein) comprising a 3- to8-amino acid oligopeptide. Metalloproteinase- and plasmin-sensitivedoxorubicin prodrugs have been developed, as well as CNGRC-peptideconjugates with doxorubicin (Hu et al. 2010, Bioorg Med Chem Lett 20,853-856; Chakravarty et al. 1983, J Med Chem 26, 638-644; Devy et al.2004, FASEB J 18, 565-567; Vanhensbergen et al. 2002, Biochem Pharmacol63, 897-908).

WO97/12624, WO97/14416, WO98/10651, WO98/18493 and WO99/02175 disclosepeptide-comprising prodrugs wherein the peptide is cleavable by theprostate-specific antigen (PSA).

Common to all above prodrugs is the presence of a protecting or cappingmoiety, usually covalently linked to the N-terminal side of theoligopeptide, which adds to the stability of the prodrug and/or adds tothe prevention of internalization of the prodrug into a cell such as atarget cell. Such protecting or capping moieties include non-naturalamino acids, β-alanyl or -succinyl groups (e.g. WO 96/05863, U.S. Pat.No. 5,962,216). Further stabilizing protecting or capping moietiesinclude diglycolic acid, maleic acid, pyroglutamic acid, glutaric acid,(e.g., WO 00/33888), a carboxylic acid, adipic acid, phthalic acid,fumaric acid, naphthalene dicarboxylic acid, 1,8-naphtyldicarboxylicacid, aconitic acid, carboxycinnamic acid, triazole dicarboxylic acid,butane disulfonic acid, polyethylene glycol (PEG) or an analog thereof(e.g., WO 01/95945), acetic acid, 1- or 2-naphthylcarboxylic acid,gluconic acid, 4-carboxyphenyl boronic acid, polyethylene glycolic acid,nipecotic acid, and isonipecotic acid (e.g., WO 02/00263, WO 02/100353),succinylated polyethylene glycol (e.g., WO 01/91798). A new type ofprotecting or capping moiety was introduced in WO 2008/120098, being a1,2,3,4 cyclobutanetetracarboxylic acid. The protecting or cappingmoiety in WO 02/07770 may be polyglutamic acid, carboxylated dextranes,carboxylated polyethylene glycol or a polymer based onhydroxyprolyl-methacrylamide or N-(2-hydroxyprolyl)methacryloylamide.

BRIEF DESCRIPTION OF THE INVENTION

The prodrugs of the invention have the general structure:

[C_(x)-OP]_(y)-D,

-   -   wherein C is a capping group;    -   OP is an oligopeptidic moiety;    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and    -   wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group, with one of the OP moieties being linked        directly, via a linker or via a spacing group to D.        Alternatively, the multiple OP moieties can each be individually        linked to D directly, via a linker, or via a spacing group. An        intermediate constellation is included wherein some of the        multiple OP moieties are individually linked to D directly, via        a linker, or via a spacing group, and wherein some of the        multiple OP moieties are themselves each individually linked to        each other directly or via a linker or spacing group, with one        of the OP moieties being linked directly, via a linker or via a        spacing group to D;    -   or a pharmaceutically acceptable salt thereof.

In particular, the oligopeptide moiety in the above structure is atetrapeptide moiety with the sequence Ala-Leu-Gly-Pro (3-letter code),also referred to as ALGP (1-letter code; SEQ ID NO:1); orAla-Leu-Ala-Leu (3-letter code), also referred to as ALAL (1-lettercode; SEQ ID NO:2), and/or the capping group C in the above structure isa phosphonoacetyl group, and/or the drug in the above structure isdoxorubicin (hereinafter also referred to as DOX or Dox). Alternatively,the structure of the tetrapeptide is ALAP (SEQ ID NO:3), TSGP (SEQ IDNO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP(SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10). Accordingly,in one embodiment the structure of the tetrapeptide in the above generalstructure is selected from the group consisting of ALGP (SEQ ID NO:1),ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ IDNO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), andKLKP (SEQ ID NO:10). In particular the tetrapeptide in the above generalstructure is selected from ALGP (SEQ ID NO: 1), or KLGP (SEQ ID NO:6);even more in particular the tetrapeptide in the above general structureis ALGP (1-letter code; SEQ ID NO:1). When present, said linker orspacing group in the above prodrugs or salts thereof may be aself-eliminating linker or spacing group.

Pharmaceutically acceptable salts of the above prodrug(s) are also partof the inventions.

The invention further relates to compositions comprising one of theabove prodrugs or salts thereof, or a combination of any thereof, and atleast one of a solvent, diluent or carrier.

The invention encompasses the above prodrug or salt thereof, or theabove composition comprising it for use in the treatment of a cancer.Methods of treating a cancer are also part of the invention, saidmethods comprising administering to a subject having cancer said prodrugor salt thereof or said composition, said administering resulting in thetreatment of said cancer. In particular, the effective amounts of saidprodrug or salt thereof, or of said composition is not causing severeleukopenia or cardiac toxicity.

Methods of producing the above prodrugs are further part of theinvention, said methods comprising the steps of:

-   -   (i) obtaining the drug;    -   (ii) linking the drug to a capped oligopeptidic moiety,        resulting in the prodrug; or, alternatively,    -   (ii′) linking the drug to an oligopeptidic moiety followed by        linking the capping group to the oligopeptidic moiety, resulting        in the prodrug; and    -   (iii) purifying the prodrug obtained in step (ii) or (ii′).

The invention further includes methods of producing and of screeningcandidate prodrugs, such candidate prodrugs having the general structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a peptide with a minimum length of 4 consecutive amino        acids (tetrapeptide) and a maximum length of 8 amino acids (i.e.        a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino        acids) which comprises carboxy-terminally a proline comprising        dipeptide selected from the group consisting of glycine-proline        (GP), alanine-proline (AP), and lysine-proline (KP); in        particular OP is a tetrapeptide with the sequence ALGP (SEQ ID        NO: 1), ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID        NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID        NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10); even more in        particular OP is the tetrapeptide ALGP (SEQ ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group and/or are individually linked to D directly or        via a linker or spacing group; and wherein said screening method        comprises the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to a GP-, AP- or KP-dipeptide to        obtain a GP-D, AP-D or KP-D as dipeptide-drug intermediate        prodrug;        (iii) contacting each of drug D and dipeptide-drug intermediate        prodrug GP-D, AP-D or KP-D independently with in vitro cultured        cells;        (iv) determining the cytotoxicity of drug D and dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D;        (v) identifying from (iv) a dipeptide-drug intermediate prodrug        GP-D, AP-D or KP-D with comparable cytotoxic activity as drug D;        and        (vi) selecting [C_(x)-OP]_(y)-D corresponding to dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D identified in step (v)        as candidate prodrug.

Alternatively, in said screening methods are methods of screeningcandidate prodrugs, the candidates to be tested have the generalstructure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a peptide with a minimum length of 4 consecutive amino        acids (tetrapeptide) and a maximum length of 8 amino acids (i.e.        a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino        acids) which comprises a carboxy-terminal proline, wherein said        proline is linked directly or via a linker or spacing group to        the drug D; in particular OP is a peptide with a minimum length        of 4 consecutive amino acids (tetrapeptide) and a maximum length        of 8 amino acids (i.e. a peptide with a length of 4, 5, 6, 7 or        8 consecutive amino acids) which comprises carboxy-terminally a        proline comprising dipeptide selected from the group consisting        of glycine-proline (GP), alanine-proline (AP), and        lysine-proline (KP); more in particular a tetrapeptide with the        sequence ALGP (SEQ ID NO: 1), ALAP (SEQ ID NO:3), TSGP (SEQ ID        NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID        NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID        NO:10); even more in particular OP is the tetrapeptide ALGP (SEQ        ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group and/or are individually linked to D directly or        via a linker or spacing group; and wherein said screening method        is comprising the steps of:

-   (i) obtaining the drug D;

-   (ii) conjugating the drug D to [C_(x)-OP]_(y) to obtain a    [C_(x)-OP]_(y)-D prodrug;

-   (iii) contacting each of drug D and prodrug [C_(x)-OP]_(y)-D    independently with in vitro cultured cells;

-   (iv) determining the cytotoxicity of drug D and prodrug    [C_(x)-OP]_(y)-D;

-   (v) identifying from (iv) a prodrug [C_(x)-OP]_(y)-D with comparable    cytotoxic activity as drug D; and

-   (vi) selecting [C_(x)-OP]_(y)-D identified in step (v) as candidate    prodrug.

In another alternative, said screening are methods of screeningcandidate prodrugs having the general structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a peptide with a minimum length of 4 consecutive amino        acids (tetrapeptide) and a maximum length of 8 amino acids (i.e.        a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino        acids) which comprises a carboxy-terminal proline, wherein said        proline is linked directly or via a linker or spacing group to        the drug D; in particular OP is a peptide with a minimum length        of 4 consecutive amino acids (tetrapeptide) and a maximum length        of 8 amino acids (i.e. a peptide with a length of 4, 5, 6, 7 or        8 consecutive amino acids) which comprises carboxy-terminally a        proline comprising dipeptide selected from the group consisting        of glycine-proline (GP), alanine-proline (AP), and        lysine-proline (KP); more in particular a tetrapeptide with the        sequence ALGP (SEQ ID NO: 1), ALAP (SEQ ID NO:3), TSGP (SEQ ID        NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID        NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID        NO:10); even more in particular OP is the tetrapeptide ALGP (SEQ        ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group and/or are individually linked to D directly or        via a linker or spacing group;

-   wherein said method is comprising the steps of:

-   (i) obtaining the drug D;

-   (ii) conjugating the drug D to [C_(x)-OP]_(y) to obtain a    [C_(x)-OP]_(y)-D prodrug;

-   (iii) contacting prodrug [C_(x)-OP]_(y)-D for 5 h at 37° C. with in    vitro cultured cells;

-   (iv) determining the conversion of prodrug [C_(x)-OP]_(y)-D into    free drug D;

-   (v) identifying from (iv) a prodrug [C_(x)-OP]_(y)-D which is    converted by at least 50% to D; and

-   (vi) selecting [C_(x)-OP]_(y)-D identified in step (v) as candidate    prodrug.

In yet another alternative, in said screening methods are methods ofscreening candidate prodrugs, the candidates to be tested have thegeneral structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a peptide with a minimum length of 4 consecutive amino        acids (tetrapeptide) and a maximum length of 8 amino acids (i.e.        a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino        acids) which comprises carboxy-terminally a proline comprising        dipeptide selected from the group consisting of glycine-proline        (GP), alanine-proline (AP), and lysine-proline (KP); more in        particular a tetrapeptide with the sequence ALGP (SEQ ID NO: 1),        ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP        (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ        ID NO:9), or KLKP (SEQ ID NO:10); even more in particular OP is        the tetrapeptide ALGP (SEQ ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group and/or are individually linked to D directly or        via a linker or spacing group; and wherein said screening method        is comprising the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to a GP-, AP- or KP-dipeptide to        obtain a GP-D, AP-D or KP-D prodrug;        (iii) contacting prodrug GP-D, AP-D or KP-D for 3 h at 37° C.        with isolated FAP and/or DPIV peptidases;        (iv) determining the conversion of prodrug GP-D, AP-D or KP-D        into free drug D;        (v) identifying from (iv) a prodrug GP-D, AP-D or KP-D which is        converted by at least 50% to D; and        (vi) selecting [C_(x)-OP]_(y)-D corresponding to prodrug GP-D,        AP-D or KP-D identified in step (v) as candidate prodrug.

Further envisaged said screening include methods of screening candidateprodrugs having the general structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a peptide with a minimum length of 4 consecutive amino        acids (tetrapeptide) and a maximum length of 8 amino acids (i.e.        a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino        acids) which comprises a carboxy-terminal proline, wherein said        proline is linked directly or via a linker or spacing group to        the drug D; in particular OP is a peptide with a minimum length        of 4 consecutive amino acids (tetrapeptide) and a maximum length        of 8 amino acids (i.e. a peptide with a length of 4, 5, 6, 7 or        8 consecutive amino acids) which comprises carboxy-terminally a        proline comprising dipeptide selected from the group consisting        of glycine-proline (GP), alanine-proline (AP), and        lysine-proline (KP); more in particular a tetrapeptide with the        sequence ALGP (SEQ ID NO: 1), ALAP (SEQ ID NO:3), TSGP (SEQ ID        NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID        NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID        NO:10); even more in particular OP is the tetrapeptide ALGP (SEQ        ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group and/or are individually linked to D directly or        via a linker or spacing group;        wherein said screening method is comprising the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to [C_(x)-OP]_(y) to obtain a        [C_(x)-OP]_(y)-D prodrug;        (iii) contacting prodrug [C_(x)-OP]_(y)-D for 3 h to 24 h at        37° C. with isolated CD10 and/or TOP peptidases and with        isolated FAP and/or DPIV peptidases;        (iv) determining the conversion of prodrug [C_(x)-OP]_(y)-D into        free drug D;        (v) identifying from (iv) a prodrug [C_(x)-OP]_(y)-D which is        converted by at least 50% to D; and        (vi) selecting [C_(x)-OP]_(y)-D identified in step (v) as        candidate prodrug.

In any of the above alternative screening methods said capping group Cmay be a phosphonoacetyl group.

In any of the above alternative screening methods said drug D may beselected from the group consisting of maytansine, geldanamycin,paclitaxel, docetaxel, campthothecin, vinblastine, vincristine,vindesine, methothrexate, aminopterin, amrubicin, or a derivative of anythereof.

In the above methods, and when present, said linker or spacing group maybe a self-eliminating linker or spacing group.

The invention further relates to kits comprising a container comprisingan above-described prodrug or salt thereof or comprising a compositioncomprising such prodrug or salt thereof.

LEGENDS TO FIGURES

FIG. 1. Effect of PhAc-peptide-Dox conjugates on the body weight of OF-1normal mice (n=4). Drugs or controls were administered by the i.v. routeon day 0. The mice receiving a constant volume (10 μL/g) of eithersaline (-•-) or of the different dosing solutions: PhAc-ALAL-Dox 80μmol/Kg (-▴-) and 160 μmol/kg (-x-); PhAc-ALGP-Dox at 240 μmol/kg (-

-) and 320 μmol/kg (-◯-). Results represent the mean body weightevolution.

FIG. 2. Efficacy study of PhAc-ALGP-Dox in comparison with freeDoxorubicin in NMRI nude mice bearing LS174T colon carcinoma humanxenografts. Drugs or controls were administered by the i.v. route on day0 and day 7. The mice receiving a constant volume (10 μL/g) of eithersaline (-•-;-∘-) or of the different dosing solutions: PhAc-ALGP-Dox 140molKg (-▴-) and 160 μmol/kg (-x-); Dox 15 μmol/kg (-♦-). Resultsrepresent the mean body weight and tumor volume evolution±SEM (n=4).

FIG. 3. Efficacy study of PhAc-ALGP-Dox in comparison with freeDoxorubicin in NMRI nude mice bearing MX-1 mammary carcinoma humanxenografts. Drugs or controls were administered by the i.v. route on day0, 3, 6 and 9. The mice receiving a constant volume (10 μL/g) of eithersaline (-•-) or of the different dosing solutions: PhAc-ALGP-Dox 100molKg (-

-); and Dox 8 μmol/kg (-▪-). Results represent the mean body weight andtumor volume evolution ±SEM (n=4).

FIG. 4. Time evolution of plasma concentration (A, B) and of cardiactissue concentration (C, D) of Doxorubicin or PhAc-ALGP-Dox and itsmetabolites after i.v. bolus injection to OF-1 female wild type mice atthe dose of 86.2 μmol/kg. Results represent the mean concentration ±SD(n=3).

FIG. 5. In vitro cellular toxicity of PhAc-ALGP-Dox and Doxorubicin onCor.At® cells (mouse embryonic stem cell derived cardiomyocytes). Asnon-specific control, the test was also performed on inactivated mouseembryonic fibroblasts (MEF). Cells were cultured for 48 h in thepresence of drugs and a neutral red uptake test was performed todetermine cell viability. Results represent the cytotoxicity curves. TheIC₅₀ values were calculated from the cytotoxicity curves using the PrismGraphPad software 5.0.

FIG. 6. PhAc-ALGP-Dox was administered at increasing concentrations (15,35, 50, 100, 200, 300, 460, 620 μmol/kg) by intravenous injection toNMRI nude mice bearing LoVo colon carcinoma xenografts (3 mice pergroup). After 24 hours, mice were sacrificed and tumors were collected.Doxorubicin tumor concentration was determined after extraction by HPLCanalysis. Results represent the average Doxorubicin tumor concentration(pmol/mg protein)±SD.

FIG. 7. Toxicity study of PhAc-ALGP-Dox after single and multipleintraperitoneal (ip) injections in OF-1 mice. Animals were treated withPhAc-ALGP-Dox with different treatment schedules: at 56 μmol/kg 5× (Q1D5-*-); at 28 μmol/kg 10× (2Q1D5 -▪-); at 560 μmol/kg 1× (-□-); at 112μmol/kg 5× (Q1D5 -▴-); at 56 μmol/kg 10× (2Q1D5 -▪-).

FIG. 8. Time evolution of blood concentration of PhAc-ALGP-Dox and itsmetabolites (A) or of Doxorubicin (B) after intraperitoneal injection toOF-1 female wild type mice at the equimolar dose of 92 μmol/kg. Resultsrepresent the mean concentration ±SD (n=3).

FIG. 9. Efficacy study of PhAc-ALGP-Dox in comparison with freeDoxorubicin in NMRI nude mice bearing LoVo colon carcinoma humanxenografts. Drugs or controls were administered by the i.p. route twicea day for five consecutive days (arrows). The mice receiving a constantvolume (10 μL/g) of either saline (-•-) or of the different dosingsolutions: PhAc-ALGP-Dox 25 μmol/kg (*), 35 μmol/kg (∘) and 50 μmol/kg(+); Dox 0.5 μmol/kg(▪), 1 μmol/kg (A) and 2 μmol/kg (x). Resultsrepresent the mean body weight and tumor volume evolution ±SEM (n=4). **Statistically different with p<0.05 from Dox 2 μmol/kg (Mann Whitney ttest of the Graph Pad Prism 5.0 software).

FIG. 10. Efficacy study of PhAc-ALGP-Dox in comparison with freeDoxorubicin in NMRI nude mice bearing MX-1 mammary carcinoma humanxenografts. Drugs or controls were administered by the i.p. route twicea day for five consecutive days (arrows). Treatment was repeated with 72hours interval between two cycles. The mice receiving a constant volume(10 μL/g) of either saline (•) or of the different dosing solutions:PhAc-ALGP-Dox 50 μmol/kg (∘), Dox 1 μmol/kg (▪), and 1.5 μmol/kg (▴).Results represent the mean body weight and tumor volume evolution ±SEM(n=6). †=dead mouse.

FIG. 11. Evaluation of PhAc-ALGP-Dox efficacy in comparison withDoxorubicin in the B16-F10 lung metastatic melanoma model. Graph on theleft side represents quantification of melanin of lung metastasis at day14 after injection of B16F10 tumor cells. Graph on the right side showthe survival curves.

FIG. 12. Evaluation of PhAc-ALGP-Dox leucopenic effect in comparisonwith Doxorubicin. CD1 mice received twice daily intraperitonealinjections of PhAc-ALGP-Dox 35 μmol/kg (•) or of Doxorubicin 3.5 μmol/kg(▪) for five consecutive days. The mice body weight evolution wasrecorded (A). Blood was collected from the tail vein in heparinisedMicrovettes tubes (Starsted) at day 4, 11 and 15 after treatmentinitiation. Peripheral white blood cells (WBC) were counted using theSCILvet abc hematologic analyzer (B). The increase or decrease in WBC isexpressed as a percentage of WBC on day 0 (100%) for each mouse. Thecurves show evolution of the mean percentages.

FIG. 13. Evaluation of chronic cardiotoxicity of PhAc-ALGP-Dox in CD-1mice. Animals received 10 intravenous injections (arrows) of saline (•);Doxorubicin 6.9 μmol/kg (▪) or of PhAc-ALGP-Dox at 13.8 (▴), 27.6 (x),55.2 (*) and 82.8 μmol/kg (∘). Results represent the evolution of theaverage relative body weight (%). †=dead mouse.

FIG. 14. Evaluation of the antitumor effect of PhAc-ALGP-dox in SCIDmice bearing orthotopic HCT116 human colon tumors. SCID mice wereinjected orthotopically (in the caecum) with human HCT116 colon tumorcells. One group was treated with saline while groups 2 and 3 received10 intraperitoneal injections (2Q1D5) of 35 and 50 μmol/kg ofPhAc-ALGP-doxorubicin. Primary tumor weights were recorded on day 34post cell inoculation.

FIG. 15. Evaluation of the antimetastatic effect (in liver) ofPhAc-ALGP-dox in SCID mice bearing orthotopic HCT116 human colon tumors.SCID mice were injected orthotopically (in the caecum) with human HCT116colon tumor cells. One group was treated with saline while groups 2 and3 received 10 intraperitoneal injections (2Q1D5) of 35 and 50 μmol/kg ofPhAc-ALGP-doxorubicin. Liver metastases were recorded on day 34 postcell inoculation.

FIG. 16. Effect of PhAc-ALGP on a UZLX-STS3 xenograft model. UZLX-STS3is a tissue xenograft derived from a patient diagnosed withdedifferentiated liposarcoma (DDLPS). During passaging, ex-mouse tumorsrevealed the same morphological and molecular features as the originalbiopsy collected from the patient during the surgery (i.e. MDM2 geneamplification, MDM2 immunopositivity). In previous xenografts not givenby perfusion this sarcoma tumor model was completely resistant to freedoxorubicin given at its maximum tolerated dose.

A total of 24 mice were bilaterally transplanted with UZLX-STS3 (tissuexenografts of passage 10). Animals were randomly assigned to threedifferent groups: a control group (saline; -•-); A PhAc-ALGP-doxorubicingroup (cumulative dose 1.20 mmol/kg; -▪-) and a doxorubicin group(cumulative dose 0.03 mmol/kg; -▴-). The drugs were administeredintraperitoneally during 7 days by continuous release via an Alzet®osmotic pump, with delivery rate of 0.5 μl/h over 7 days. The experimentlasted 21 days (7 days of treatment+14 days of observation). Tumorvolume and mouse body weight were evaluated at baseline and subsequentlythree times per week until the end of each experiment. After 21 daysmice were sacrificed. Tumor volume is recorded 3× weekly by3-dimensional caliper measurement. Data are presented as the average ofthe relative tumor volumes per group ±standard deviation.

The comparison between the tumor volumes on day zero and the volumes onthe last day of the experiments was done by Wilcoxon's matched pairedtest. The comparisons between different groups were performed using theMann-Whitney U test (relative tumor volumes, histologic assessment).P<0.05 was considered as statistically significantly different. TheSTATISTICA software (StatSoft, version 12.0) was used for all thecalculations.

FIG. 17. Relative body weight evolution of the animals of the experimentshown in FIG. 16. Data are presented as the average of the relative bodyweight of mice in every group±standard deviation. The dashed lines markthe reference values for nu/nu NMRI Mouse strain. PhAc-ALGP-doxorubicin(-▪-); Dox (-▴-); and saline (-•-).

FIG. 18. Total white blood cell count (10³/μL) evolution of the animalsof the experiment shown in FIG. 16. Data are presented as the average ofthe white blood cell count of mice in every group ±standard deviation.The dashed lines mark the reference values for nu/nu NMRI mouse strain.PhAc-ALGP-doxorubicin (-▪-); Dox (-▴-); and saline (-•-). Total whiteblood cell count was determined using the CELL-DYN 3500 multiparameterautomated hematology analyzer with optimization for murine bloodparameters (Abott Diagnostics, Division, IL, USA) FIG. 19. Totalneutrophil count (10³/μL) evolution of the animals of the experimentshown in FIG. 16. Data are presented as the average of the neutrophilcount of mice in every group ±standard deviation. The dashed lines markthe reference values for nu/nu NMRI mouse strain. PhAc-ALGP-doxorubicin(-▪-); Dox (-▴-); and saline (-•-). Neutrophil count was determinedusing the CELL-DYN 3500 multiparameter automated hematology analyzerwith optimization for murine blood parameters (Abott Diagnostics,Division, IL, USA)

DETAILED DESCRIPTION OF THE INVENTION

In general, the present invention describes new prodrug compounds oftherapeutic agents, especially prodrugs comprising an antitumortherapeutic agent, displaying improved therapeutic properties. Theimproved therapeutic properties include decreased toxicity and increasedefficacy. In particular, the prodrugs display a high specificity ofaction, a reduced toxicity, an improved stability in the serum andblood, and their therapeutic agent is not moving into target cells untilthe prodrug is finally activated (the activation may involve multiplesteps) by (a) target cell associated enzyme(s) such as extracellularlypeptidases released from the target cells or such as associated with theextracellular surface of the target cells. Target cells include cancercells as well as tumor stroma cells. The prodrug compounds of theinvention are prodrug forms of a therapeutic agent, in which thetherapeutic agent is linked directly or indirectly to an oligopeptide,which in turn, is linked to a stabilizing capping group.

In general, the prodrugs of the invention have the following generalstructure:

[C_(x)-OP]_(y)-D,

-   -   wherein C is a capping group;        -   OP is an oligopeptidic moiety;        -   D is a drug;        -   x is an integer being at least 1 when y=1;        -   y is an integer being at least 1, if y is greater than 1,            then at least 1 OP is carrying a capping group; and    -   wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group, with one of the OP moieties being linked        directly, via a linker or via a spacing group to D. In other        words, multiple OP moieties may be linked together to form a        linear or branched structure which is linked to D via one of the        OP moieties in the linear or branched structure. Alternatively,        the multiple OP moieties are each individually linked to D        directly, via a linker, or via a spacing group. An intermediate        constellation is included wherein some of the multiple OP        moieties are each individually linked to D directly, via a        linker, or via a spacing group, and wherein some of the multiple        OP moieties are themselves individually linked to each other as        described above to form a linear or branched structure of which        one of the OP moieties being linked directly, via a linker or        via a spacing group to D;    -   or a pharmaceutically acceptable salt thereof.

For clarification, and without being exhaustive, when y=2 the followingprodrugs are included in the general structure: C-OP-D-OP-C; C—OP-OP-Dand C-OP-D-OP. The capping group C thus can be present (via direct orindirect linkage as described above) on one or more oligopeptidemoieties OP in the case of a prodrug compound/molecule comprisingmultiple oligopeptide moieties OP.

In one embodiment, the oligopeptidic moiety is a peptide with a minimumlength of 4 consecutive amino acids (tetrapeptide) and a maximum lengthof 8 amino acids (i.e. a peptide with a length of 4, 5, 6, 7 or 8consecutive amino acids) which comprises a carboxy-terminal proline,wherein said proline is linked directly or via a linker or spacing groupto the drug D.

Without being limited thereto, it is our understanding that thisoligopeptidic moiety is cleaved/removed from the drug D in a two-stepprocess wherein the first step converts said prodrug to a dipeptide-drugintermediate and wherein the second step converts the dipeptide-drugintermediate to the free drug D. In a particular embodiment thedipeptide remaining in the the dipeptide-drug intermediate has thesequence glycine-proline (GP), alanine-proline (AP), or lysine-proline(KP). It is thus an object of the present invention to provide theprodrugs with the above-mentioned general formula, wherein OP representsa peptide with a minimum length of 4 consecutive amino acids(tetrapeptide) and a maximum length of 8 amino acids (i.e. a peptidewith a length of 4, 5, 6, 7 or 8 consecutive amino acids) whichcomprises carboxy-terminally a proline comprising dipeptide selectedfrom the group consisting of glycine-proline (GP), alanine-proline (AP),and lysine-proline (KP), wherein the proline of said proline comprisingdipeptide is linked directly or via a linker or spacing group to thedrug D.

In particular, the oligopeptide moiety in the above structure is atetrapeptide moiety with the sequence Ala-Leu-Gly-Pro (3-letter code),also referred to as ALGP (1-letter code; SEQ ID NO:1); orAla-Leu-Ala-Leu (3-letter code), also referred to as ALAL (1-lettercode; SEQ ID NO:2), and/or the capping group C in the above structure isa phosphonoacetyl group, and/or the drug in the above structure isdoxorubicin (hereinafter also referred to as DOX or Dox). Alternatively,the structure of the tetrapeptide is ALAP (SEQ ID NO:3), TSGP (SEQ IDNO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP(SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO:10). Accordingly,in one embodiment the structure of the tetrapeptide in the above generalstructure is selected from the group consisting of ALGP (SEQ ID NO:1),ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ IDNO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), andKLKP (SEQ ID NO:10). In particular the tetrapeptide in the above generalstructure is selected from ALGP (SEQ ID NO: 1), or KLGP (SEQ ID NO:6);even more in particular the tetrapeptide in the above general structureis ALGP (1-letter code; SEQ ID NO: 1). In a preferred embodiment of thepresent invention the drug (D) in the above general structure isdoxorubicin or a pharmaceutically acceptable salt thereof.

In particular, the prodrug may have the structure of Compound I (seeExample 3.1) or may be a pharmaceutically acceptable salt thereof. Thecapping group phosphonoacetyl provides the advantages of avoiding theuse of a non natural amino acids at the terminal end of theoligopeptide. When the drug D is doxorubicin, phosphonoacetyl as cappinggroup C has the further advantage of providing a negatively charge thatis important in order to avoid aggregation of oligopeptide derivativesof doxorubicin. In general, the stability of a prodrug of the inventioncan be defined such that less than 10% of cleavage derivatives are to beobtained upon incubation of the prodrug in human blood for more than 2hours.

Notwithstanding the above definitions of the capping group C, theoligopeptide moiety OP and the drug, these are not limiting the currentinvention and other combinations are envisaged by the invention. Thesecombinations include any OP and/or D with the capping group C being aphosphonoacetyl group. Further combinations include any C (any cappinggroup known in the art) and/or any D with the oligopeptide moiety OPbeing a tetrapeptide moiety with the sequence Ala-Leu-Gly-Pro (3-lettercode) or Ala-Leu-Ala-Leu (3-letter code) or ALGP (1-letter code; SEQ IDNO:1) or ALAL (1-letter code; SEQ ID NO:2). Alternatively, the structureof the tetrapeptide is ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQID NO:5), KLGP (SEQ ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8),TSKP (SEQ ID NO:9), or KLKP (SEQ ID NO: 10). In a further alternativethe structure of the tetrapeptide is ALGP (SEQ ID NO:1), ALAP (SEQ IDNO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ ID NO:6), KLAP(SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), or KLKP (SEQ IDNO: 10). In particular the tetrapeptide in the above general structureis selected from ALGP (SEQ ID NO: 1), or KLGP (SEQ ID NO:6); even morein particular the tetrapeptide in the above general structure is ALGP(1-letter code; SEQ ID NO: 1).

Depending on the structure/chemical formula of the drug, 1 or more ofthe oligopeptidic moieties of the invention, of which at least one ofthem is capped, can be linked to the drug. The oligopeptidic moietiescan themselves form a linear or branched structure linked to the drugor, alternatively, multiple oligopeptidic moieties are each individuallylinked to the drug.

The capping group or protecting or capping moiety is linked to theoligopeptidic moiety of the prodrug and adds to the solubility and/orstability of the prodrug (e.g. in blood of the animal, mammal, human orsubject to which the prodrug is administered) and/or adds to theprevention of internalization of the prodrug into a cell such as atarget cell.

The linkage between the capping group and the oligopeptide and/orbetween the oligopeptide and the therapeutic agent or drug may bedirect, e.g. via the N-terminal aminogroup of the oligopeptide or theC-terminal carboxylgroup of the oligopeptide, or via a side chain of oneof the amino acids of the oligopeptide.

Alternatively, said linkage may be indirect, e.g. by introducing alinker or spacer group between the oligopeptide OP and the drug D. Inthe case of cytotoxic compounds such as doxorubicin having a free amino(NH₂) group, a linker between D and OP is not required per se asenzymatic scission of the amide bond between D and OP ensures theavailability of the free NH₂-group on the cytoxic drug.

However most of the anticancer cytotoxic drugs D do not have any freeNH₂-group and cannot as such be linked to OP by an amide bond.Introducing an NH₂-group to those molecules may decrease or suppresstheir cytotoxic activity. For such drugs, a self-immolating (orself-eliminating, self-sacrificing, self-lysing, or self-leaving)spacing group or spacer can be used as linker between the drug D and theoligopeptide OP. OP is linked to the self-immolating spacer by an amidebond sensitive to extracellular enzymes capable of activating theprodrug. After cleavage of the amide bond between OP and the spacer, theself-immolating spacer cleaves itself from the drug leaving itunderivatized, i.e., leaving it in its original active form.Self-immolative spacers include para-aminobenzoyloxycarbonyl moietiesthat are able to connect either —OH, —COOH, —NH, or —SH groups of a drugat the one hand to the carboxy-terminal group of a peptide at the otherhand. This type of linker is an electronic cascade spacer. Such bond hasbeen shown to be cleavable by peptidases. After cleavage of theOP-spacer amide bond, the aromatic amine of the self-eliminating spacerbecomes electron-donating, which leads to expulsion and release of thefree drug and CO₂ (Carl et al. 1981, J Med Chem 24, 479-480; Chakravartyet al. 1983, J Med Chem 26, 638-644; de Groot et al. 1999, J Med Chem42, 5277-5283, King et al. 2002, J Med Chem 45, 4336-4343). Severalpatents and patent applications describe otherself-immolative/self-eliminating spacers, such as heterocyclic ones,releasing a drug from a targeting ligand such as an antibody have beendescribed (e.g. U.S. Pat. No. 6,214,345; US 2003/0130189; US2003/0096743; U.S. Pat. No. 6,759,509; US 2004/0052793; U.S. Pat. Nos.6,218,519; 6,835,807; 6,268,488; US 2004/0018194; WO 98/13059; US2004/0052793; U.S. Pat. Nos. 6,677,435; 5,621,002; US 2004/0121940; WO2004/032828, US 2009/0041791).

Examples of other, not necessarily self-eliminating, linker or spacergroups include aminocaproic acid, a hydrazide group, en ester group, anether group and a sulphydryl group. A linker or spacer group asdescribed above between the capping group and the oligopeptide and/orbetween the oligopeptide and the therapeutic agent may be advantageousfor reasons such as the following: (i) as a spacer for stericconsiderations in order to facilitate enzymatic release of the aminoacid linked to the therapeutic agent or other enzymatic activationsteps; (ii) to provide an appropriate attachment chemistry between thedifferent moieties of the prodrug (and thus providing flexibility tocouple any possible drug and/or capping moiety to the oligopeptide ofthe invention); (iii) to improve the synthetic process of making theprodrug conjugate (e.g., by pre-derivatizing the therapeutic agent oroligopeptide with the linker group before conjugation to enhance yieldor specificity); (iv) to improve physical properties of the prodrug; or(v) to provide an additional mechanism for intracellular release of thedrug. Whatever the type of linkage, direct or indirect, the linkageshould: (1) not or not significantly disturb the functionality of theoligopeptidic moiety, i.e., should not significantly disturb neither theproteolytic scissability by TOP nor the resistance to proteolyticscissability by CD1 and (2) retain the blood stability of the compound.Determination of the functionality of the capped oligopeptidic moiety inthe prodrug can be tested easily and in a straightforward way, e.g. asdescribed in the Examples section hereafter. Such testing does notamount to an undue burden for a skilled person.

The phrase “pharmaceutically acceptable salt(s)”, as used herein, meansthose salts of compounds of the invention that are safe and effectivefor the intended medical use that possess the desired biologicalactivity. Pharmaceutically acceptable salts include salts of acidic orbasic groups present in compounds of the invention. Suitable base saltsinclude, but are not limited to, aluminum, calcium, lithium, magnesium,potassium, sodium, zinc, and diethanolamine salts. For a review onpharmaceutically acceptable salts see, e.g., Berge et al. 1977 (J.Pharm. Sci. 66, 1-19) or Handbook of Pharmaceutical Salts: Properties,Selection, and Use (P. H. Stahl, C. G. Wermuth (Eds.), August 2002),incorporated herein by reference.

The drug or therapeutic agent conjugated to the oligopeptide of theinvention may be useful for treatment of cancer (e.g. by exertingcytotoxic or antiangiogenic activity), inflammatory disease, or someother medical condition. The drug or therapeutic agent conjugated to theoligopeptide of the invention may be any drug or therapeutic agentcapable of entering a target cell. Thus, the therapeutic agent may beselected from a number of classes of compounds including, antibiotics,alkylating agents, antiproliferative agents, tubulin binding agents,vinca alkaloids, enediynes, podophyllotoxins or podophyllotoxinderivatives, the pteridine family of drugs, taxanes, anthracyclines,dolastatins, topoisomerase inhibitors, platinum-coordination-complexchemotherapeutic agents, and maytansinoids. More in particular, saiddrug or therapeutic agent may be one of the following compounds, or aderivative or analog thereof: doxorubicin, daunorubicin, amrubicin,vinblastine, vincristine, calicheamicin, etoposide, etoposide phosphate,CC-1065, duocarmycin, KW-2189, methotrexate, methopterin, aminopterin,dichloromethotrexate, docetaxel, paclitaxel, epithiolone,combretastatin, combretastatin A4 phosphate, dolastatin 10. dolastatin11, dolastatin 15, topotecan, camptothecin, mitomycin C, porfiromycin, 5fluorouracil, 6-mercaptopurine, fludarabine, tamoxifen, cytosinearabinoside, adenosine arabinoside, colchicine, halichondrin B,cisplatin, carboplatin, mitomycin C, bleomycin, melphalan, chloroquine,cyclosporin A, and maytansine. By derivative is intended a compound thatresults from reacting the named compound with another chemical moiety(different from the oligopeptidic moiety linked directly or indirectlyto the compound), and includes a pharmaceutically acceptable salt, acid,base or ester of the named compound. Other therapeutic agents or drugsinclude: vindesine, vinorelbine, 10-deacetyltaxol, 7-epi-taxol, baccatinIII, 7-xylosyltaxol, isotaxel, ifosfamide, chloroaminophene,procarbazine, chlorambucil, thiophosphoramide, busulfan, dacarbazine(DT1C), geldanamycin, nitroso ureas, estramustine, BCNU, CCNU,fotemustine, streptonigrin, oxaliplatin, methotrexate, aminopterin,raltitrexed, gemcitabine, cladribine, clofarabine, pentostatin,hydroxyureas, irinotecan, topotecan,9-dimethylaminomethyl-hydroxy-camptothecin hydrochloride, teniposide,amsacrine; mitoxantrone; L-canavanine, THP-adriamycin, idarubicin,rubidazone, pirarubicin, zorubicin, aclarubicin, epiadriamycin(4′epi-adriamycin or epirubicin), mitoxantrone, bleomycins, actinomycinsincluding actinomycin D, streptozotocin, calicheamycin; L-asparaginase;hormones; pure inhibitors of aromatase; androgens, proteasomeinhibitors; farnesyl-transferase inhibitors (FTI); epothilones;discodermolide; fostriecin; inhibitors of tyrosine kinases such as STI571 (imatinib mesylate); receptor tyrosine kinase inhibitors such aserlotinib, sorafenib, vandetanib, canertinib, PKI 166, gefitinib,sunitinib, lapatinib, EKB-569; Bcr-Abl kinase inhibitors such asdasatinib, nilotinib, imatinib; aurora kinase inhibitors such as VX-680,CYC116, PHA-739358, SU-6668, JNJ-7706621, MLN8054, AZD-1152, PHA-680632;CDK inhibitors such as flavopirodol, seliciclib, E7070, BMS-387032; MEKinhibitors such as PD184352, U-0126; mTOR inhibitors such as CCI-779 orAP23573; kinesin spindle inhibitors such as ispinesib or MK-0731;RAF/MEK inhibitors such as Sorafenib, CHIR-265, PLX-4032, CI-1040,PD0325901 or ARRY-142886; bryostatin; L-779450; LY333531; endostatins;the HSP 90 binding agent geldanamycin, macrocyclic polyethers such ashalichondrin B, eribulin. For a number of compounds included in theabove listing, more experimental guidance is given in Example 16 herein.Amongst the drugs other than doxorubicin covered in this invention isamrubicin which is an anthracycline analogue with a free NH₂-group(Hanada et al. 1998, Jpn J Cancer Res 89, 1229-1238) that can be linkedto a capped oligopeptide such as PhAc-ALGP by the same method as usedfor doxorubicin. Addition of (a) polyethylene glycol group(s) to theamino- of the oligopeptidic moiety) may be performed in order toincrease the half-life of the prodrug according to the invention incirculation after administration to a mammal. Addition of (a)polyethylene glycol group(s) could also play the role of a cappingagent.

A prodrug or salt thereof of the invention can further be present in acomposition comprising besides the prodrug or salt thereof any one of asuitable solvent (capable of solubilizing the prodrug to the desiredextent), diluent (capable of diluting concentrated prodrug to thedesired extent) or carrier (any compound capable of absorbing, adheringor incorporating the prodrug, and of subsequently releasing at any ratethe prodrug in the extracellular compartment of the subject's body).Said composition may alternatively comprise multiple (i.e. more than 1)prodrug or salt thereof, or any combination thereof (e.g. prodrug 1+itssalt, prodrug 1+prodrug 2, prodrug 1+its salt+prodrug 2, etc.) Inparticular, said solvent, diluents or carrier is pharmaceuticallyacceptable, i.e., is acceptable to be administered to a subject to betreated with the composition of the invention. Aiding in formulating apharmaceutically acceptable composition is e.g. any Pharmacopeia book.The composition may be formulated such that it is suitable for any wayof administration including intra-cranial, intra-spinal, enteral andparenteral administration. The regimen by which the prodrug isadministered may vary, e.g. depending on whether or not a capping groupis present, depending on the formulation, depending on the overallphysical condition of a subject to be treated and e.g. depending on thejudgment of the treating physician.

The prodrug or salt thereof of the invention, or a compositioncomprising it, is particularly suitable for treating a disease that istreatable by the released drug. Of particular interest is cancer ortumors such as solid tumors. “Cancer” includes e.g. breast cancers,colorectal cancers, liver cancers, lung cancers such as small cell,non-small cell, bronchic cancers, prostate cancers, ovarian cancers,brain cancers, and pancreatic cancers, colon cancers, head and neckcancers, stomach cancers, bladder cancers, non-Hodgkin's lymphomas,melanomas, leukaemias, neuroblastomas, and glioblastomas. The subject tobe treated with the prodrug of the invention can be any mammal in needof such treatment but is in particular a human. The treatment can resultin regression of the disease (e.g. in terms of decreasing tumor volumeor tumor mass and of metastases), in decreased progression of thedisease compared to expected disease progression, or in stabilization ofthe disease, i.e. neither regression nor progression of the disease. Allthese are favorable outcomes of the treatment. In particular, theeffective amounts of said prodrug or salt thereof, or of saidcomposition is not causing severe leukopenia or cardiactoxicity/cardiotoxicity. A possible definition of severe humanleukopenia is WHO-criteria-defined grade 3-(1000-1900 leukocytes/mL) orgrade 4-leukopenia (less than 1000 leukocytes/mL).

Inclusion of an anticancer prodrug (or a salt thereof) according to thepresent invention in combination therapies is also envisaged. This canbe in a combined modality chemotherapy, i.e. the use of the anticancerprodrug (or a salt thereof) with other cancer treatments, such asradiation therapy (whether by direct irradiation or via administering anisotope-labeled antibody or antibody fragment) or surgery. This can alsobe in combination chemotherapy, i.e. treating a patient with a number ofdifferent drugs wherein the drugs preferably differ in their mechanismof action and in their side effects. Usually in such combinationchemotherapy the drugs are administered simultaneously. An advantage ofcombination chemotherapy is the minimization of the chance of thedevelopment of resistance to any one agent. A further advantage may bethat the individual drugs can each be used at a lower dose, therebyreducing overall toxicity.

A prodrug or salt thereof according to the invention, or a compositioncomprising such prodrug or salt, can thus be used for treatment of adisease (e.g. cancer), as monotherapy, or as part of a combinationchemotherapy treatment or a combined modality chemotherapy treatment.

More in general in relation to combination chemotherapy, an anticancerprodrug (or a salt thereof) according to the invention can be combinedwith one or more alkylating antineoplastic agent(s) and/or one or moreanti-metabolite(s) and/or one or more anti-microtubule agent(s) and/orone or more topoisomerase inhibitor(s) and/or one or more cytotoxicantibiotic(s) and/or one or more biological anticancer agent(s) (such asantibodies), wherein one or more of these, when applicable, can also beprodrug(s) (or a salt thereof) according to the present invention.

The drug doxorubicin (also known under the trade names Adriamycin orRubex) is commonly used to treat multiple types of cancers such as someleukemias and Hodgkin's lymphoma, as well as cancers of the bladder,breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiplemyeloma, and others. Doxorubicin is further used in differentcombination therapies. Doxorubicin-containing therapies include AC or CA(Adriamycin, cyclophosphamide), TAC (Taxotere, AC), ABVD (Adriamycin,bleomycin, vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide,Adriamycin (doxorubicin), cyclophosphamide, Oncovin (vincristine),procarbazine, prednisone), CHOP (cyclophosphamide, Adriamycin,vincristine, prednisolone), FAC or CAF (5-fluorouracil, Adriamycin,cyclophosphamide), MVAC (methothrexate, vincristine, adriamycin,cisplatin), CAV (cyclophosphamide, doxorubicin, vincristine) and CAVE(CAV, etoposide), CVAD (cyclophosphamide, vincristine, adriamycin,dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin orplatinol, adriamycin, cyclophosphamide, etoposide), m-BACOD(methothrexate, bleomycin, adriamycin, cyclophosphamide, vincristine,dexamethasone), MACOP-B (methothrexate, leucovorin, adriamycin,cyclophosphamide, vincristine, prednisone, bleomycin), Pro-MACE-MOPP(methothrexate, adriamycin, cyclophosphamide, etoposide,mechlorethamine, vincristine, procarbazine, prednisone), ProMACE-CytaBOM(prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine,bleomycin, vincristine, methothrexate, leucovorin), Stanford V(doxorubicin, mechlorethamine, bleomycin, vinblastine, vincristine,etoposide, prednisone), DD-4A (vincristine, actinomycin, doxorubicin),VAD (vincristine, doxorubicin, dexamethasone), Regimen I (vincristine,doxorubicin, etoposide, cyclophosphamide) and VAPEC-B (vincristine,doxorubicin, prednisone, etoposide, cyclophosphamide, bleomycin).Besides the doxorubicin-comprising combination chemotherapies there is aplethora of other combination chemotherapies such as BEP (Bleomycin,etoposide, platinum agent (cisplatin (Platinol))), CAPOX or XELOX(capecitabine, oxaliplatin), CBV (cyclophosphamide, carmustine,etoposide), FOLFIRI (fluorouracil, leucovorin, irinotecan), FOLFIRINOX(fluorouracil, leucovorin, irinotecan, oxaliplatin), FOLFOX(fluorouracil, leucovorin, oxaliplatin), EC (epirubicin,cyclophosphamide), ICE (ifosfamide, carboplatin, etoposide (VP-16)) andIFL (irinotecan, leucovorin, fluorouracil). Combination of doxorubicinwith sirolimus (rapamycin) has been disclosed by Wendel et al. 2004(Nature 428, 332-337) in treatment of Akt-positive lymphomas in mice. Inany of these combination therapies any of the drugs could be substitutedby a prodrug (or a salt thereof) according to the present invention.

One can further also envisage combination therapies including ananticancer prodrug (or a salt thereof) according to the invention(whether alone or already part of a combination chemotherapy or of acombined modality therapy) and compounds other than cytostatics. Suchother compounds include any compound approved for treating cancer orbeing developed for treating cancer. In particular, such other compoundsinclude monoclonal antibodies such as alemtuzumab (chronic lymphocyticleukemia), bevacizumab (colorectal cancer), cetuximab (colorectalcancer, head and neck cancer), denosumab (solid tumor's bonymetastases), gemtuzumab (acute myelogenous leukemia), ipilimumab(melanoma), ofatumumab (chronic lymphocytic leukemia), panitumumab(colorectal cancer), rituximab (Non-Hodgkin lymphoma), tositumomab(Non-Hodgkin lymphoma) and trastuzumab (breast cancer). Other antibodiesinclude for instance abagovomab (ovarian cancer), adecatumumab (prostateand breast cancer), afutuzumab (lymphoma), amatuximab, apolizumab(hematological cancers), blinatumomab, cixutumumab (solid tumors),dacetuzumab (hematologic cancers), elotuzumab (multiple myeloma),farletuzumab (ovarian cancer), intetumumab (solid tumors), Matuzumab(colorectal, lung and stomach cancer), onartuzumab, parsatuzumab,pritumumab (brain cancer), tremelimumab, ublituximab, veltuzumab(non-Hodgkin's lymphoma), votumumab (colorectal tumors), zatuximab andanti-placental growth factor antibodies such as described in WO2006/099698. Examples of such combination therapies include for instanceCHOP-R (CHOP (see above)+rituximab), ICE-R (ICE (see above)+rituximab),R-FCM (rituximab, fludarabine, cyclophosphamide, mitoxantrone) and TCH(Paclitaxel (Taxol), carboplatin, trastuzumab).

Examples of alkylating antineoplastic agents include nitrogen mustards(for example mechlorethamine, cyclophosphamide, melphalan, chlorambucil,ifosfamide and busulfan), nitrosoureas (for exampleN-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU),semustine (MeCCNU), fotemustine and streptozotocin), tetrazines (forexample dacarbazine, mitozolomide and temozolomide), aziridines (forexample thiotepa, mytomycin and diaziquone (AZQ)), cisplatins andderivatives (for example cisplatin, carboplatin and oxaliplatin), andnon-classical alkylating agents (for example procarbazine andhexamethylmelamine)

Subtypes of the anti-metabolites include the anti-folates (for examplemethotrexate and pemetrexed), fluoropyrimidines (for examplefluorouracil, capecitabine and tegafur/uracil), deoxynucleosideanalogues (for example cytarabine, gemcitabine, decitabine, Vidaza,fludarabine, nelarabine, cladribine, clofarabine and pentostatin) andthiopurines (for example thioguanine and mercaptopurine).

Anti-microtubule agents include the vinca alkaloid subtypes (for examplevincristine, vinblastine, vinorelbine, vindesine and vinflunine) andtaxane subtypes (for example paclitaxel and docetaxel). Otheranti-microtubule agents include podophyllotoxin.

Topoisomerase inhibitors include topoisomerase I inhibitors (for exampleirinotecan, topotecan and camptothecin) and topoisomerase II inhibitors(for example etoposide, doxorubicin, mitoxantrone, teniposide,novobiocin, merbarone, and aclarubicin).

Cytotoxic antibiotics include anthracyclines (doxorubicin, daunorubicin,epirubicin, idarubicin, pirarubicin, aclarubicin and mitoxantrone) andother drugs including actinomycin, bleomycin, plicamycin and mitomycin.

Any anticancer prodrug (or a salt thereof) according to the inventioncan (whether alone or already part of a combination chemotherapy or of acombined modality therapy) further be included in an antibody-directedenzyme prodrug therapy (ADEPT), which includes the application ofcancer-associated monoclonal antibodies, which are linked, to adrug-activating enzyme. Subsequent systemic administration of anon-toxic agent results in its conversion to a toxic drug, and resultingin a cytotoxic effect which can be targeted at malignant cells (Bagshaweet al. (1995) Tumor Targeting 1, 17-29.)

Further, any anticancer prodrug (or a salt thereof) according to theinvention can (whether alone or already part of a combinationchemotherapy or of a combined modality therapy) be combined with one ormore agent(s) capable of reversing (multi)drug resistance ((M)DRreverser(s) or (M)DR reversing agent(s)) that can occur duringchemotherapy. Such agents include for example loperamide (Zhou et al.2011, Cancer Invest 30, 119-125). Another such combination includesloading the prodrug in nanoparticles such as iron oxide nanoparticles(Kievit et al. 2011, J Control Release 152, 76-83) or liposomes.Examples of drugs loaded into liposomes include doxorubicin (doxorubicinHCL liposomes, also known under the trade names Doxil, Caelyx orMyocet), daunorubicin (known under the trade name DaunoXome) andpaclitaxel (Garcion et al. 2006, Mol Cancer Ther 5, 1710-1722).

A prodrug or salt thereof according to the invention, or a compositioncomprising such prodrug or salt, can thus be used for treatment of adisease (e.g. cancer), as monotherapy, or as part of a combinationchemotherapy treatment or a combined modality chemotherapy treatment.Any of such treatments can further be combined with a treatmentincluding a drug resistance reverting agent.

The invention further relates to methods of producing the prodrugs ofthe invention, said methods comprising the steps of:

-   -   (i) obtaining the drug;    -   (ii) linking the drug to a phosphonoacetyl-capped oligopeptidic        moiety, resulting in the prodrug; or, alternatively,    -   (ii′) linking the drug to an oligopeptidic moiety followed by        linking the phosphonoacetyl capping group to the oligopeptidic        moiety, resulting in the prodrug; and    -   (iii) purifying the prodrug obtained in step (ii) or (ii′).

As described above, said linking of the oligopeptidic moiety with thedrug and/or capping group may be direct, or indirect via a linker orspacing group, such as a self-immolating or self-eliminating spacer. Thepurification strategy of the prodrug will obviously depend on the natureof the drug and/or of the capping group. A skilled person will be ableto design a suitable purification strategy for any possible prodrugaccording to the invention, choosing from a plethora of purificationtechniques that are available.

Without being bound by any theory or explanation, the picture emergingfrom the Examples as described herein is one that, for the exemplaryprodrug of the invention comprising the ALGP-peptide (SEQ ID NO:1) as OPmoiety and doxorubicin as the drug D, the activation of the prodrug isoccurring in multiple steps. Whereas such prodrug is stable in blood andplasma, it is converted in a mixture of doxorubicin (Dox), GP-Dox andLGP-Dox when incubated in the presence of LS-174T tumor cells. Thelatter process can in a first step be mimicked in vitro by proteasessuch as CD10 (yielding LGP-Dox that can be converted to GP-Dox by commonleucine aminopeptidases) and TOP (yielding GP-Dox). The first phase ofthe activation of the ALGP-doxorubicin is driven by the preferentialactivity of CD10 and TOP in the vicinity of the tumors compared to theirlower abundancy in non-pathological extracellular compartments andtissues. The second step, conversion of GP-Dox to Dox, can be driven bydipeptidyl prolyl peptidases. Two members of this class are of interestin the area of cancer: DPIV, also known as CD26 and FAP or fibroblastactivation protein. All these proteases are known to be associated withtumor cells or tumor stromal cells as described hereafter. Suchmultistep activation of a prodrug of the invention increases thespecificity and decreases the unwanted side effects (such as leucopeniaand cardiac toxicity) compared to similar prodrugs that are activatablein a single step. An example of the latter is succinyl-βALAL-doxorubicinwhich is easily converted by e.g. CD10 to L-Dox that can enter the cellon its own (Pan et al. 2003, Cancer Res 63, 5526-5531). The multipleactivation steps approach yielded a PhAc-ALGP-doxorubicin prodrug thatis about 20 to 40 times less toxic than doxorubicin varying with themode (IV or IP) of administration, and between 6 and 14 times less toxicthan succinyl-β-ALAL-doxorubicin. PhAc-ALGP-doxorubicin is devoid ofchronic cumulative cardiotoxicity and does not induce leucopenia andlymphopenia. It is more active than doxorubicin on human tumorxenografts (including a sarcoma) and on an orthotopic colon carcinoma.The prodrugs of the invention therefore are further characterized bybeing activatable, in vitro or in vivo, in at least two steps, i.e., ina process involving at least two essential proteolytic cleavages by atleast two different proteases. An “essential proteolytic cleavage” isherein meant to be a cleavage that is associated with a tumor or atumor-associated cell such as its stroma, i.e., is specificallyoccurring in the direct vicinity of a tumor or tumor-associated cells.

Endopeptidases CD10 and TOP

CD10 is a neutral endopeptidase/a zinc dependent cell surfacemetallopeptidase that cleaves small peptides on the amino-side ofhydrophobic amino acids. Besides being present on normal cells such as Bcells and epithelial cells of the lung, colon and kidney, it is presentin many tumor types (Ravel et al. 2008, Clin Cancer Res 14, 1258-1265),such as pancreatic cancer (Notohara et al. 2000, Am J Surg Pathol 24,1361-1371), hepatocellular carcinoma (Karabork et al. 2010, Pathol ResPract 206, 572-577), melanoma (Velazquez et al. 2007, J Transl Med 5,2), prostate cancer (Song et al. 2004, Prostate 58, 394-405), lung smallcell carcinomas (Shipp et al. 1991, Proc Natl Acad Sci USA 88,10662-10666), renal carcinoma, endometrial sarcoma and rhabdomyosarcoma(Chu et al. 2000, Am J Clin Pathol 113, 374-382). CD10 is expressed innearly half of the soft tissue sarcomas (histiocytomas, fibrosarcomas,rhabdomyosarcomas, leiomyosarcomas, liposarcomas, malignant peripheralnerve sheat tumors; Deniz et al. 2012, Pathol Res Pract 208, 281-285).Even more interesting and similar to the stromal distribution of FAP,CD10 is found in the stromal cells of colorectal carcinomas (Hirano etal. 2012, Pathol Int 62,600-611), breast cancer (Desmedt et al. 2012,Clin Cancer Res 18, 1004-1014; Marketsov et al. 20, 84-89), pancreaticendocrine tumors (Deschamps et al. 2006, Hum Path 37, 802-808), gastriccarcinoma (Huang et al. 2005, Jpn J Clin Oncol 35, 245-250) and basalcell carcinoma (Yada et al. 2004, Am J Dermatopathol 26, 463-473).

TOP (Thimet Oligo Peptidase) is a thiol-dependent main cytoplasmicmetallo-endoprotease distributed throughout many tissues and cell types.It can attain an extracellular location both via secretion of thesoluble enzyme and by attachment to the plasma membrane. It isdistributed throughout many tissues and cell types. TOP is involved inneuroendocrine signaling and in the extracellular metabolism ofneuropeptides (Corie et al. 2002, Endocrine Rev 23, 647-664). It isinvolved in the metabolism of proteasomes (Saric et al. 2004, J BiolChem 45, 46723-46732). TOP is involved in neuropeptide processing inprostate and prostate cancer (Swanson et al. 2004, Protein Pept Lett 5,471-478) and is found in tumor cell conditioned media. It can bereleased from damaged or necrotic cells. Its activity is reduced inoxygenated media and enhanced in anoxic environments that are very oftencharacteristic of solid tumors. TOP is expressed at the surface ofendothelial cells and plays a role in vasoactive peptide metabolism(Norman et al. 2003, Am J Physiol Heart Circ Physiol 284, H1978-1984;Shivakumar et al. 2005, Cell Biochem Funct 23, 195-204). Top is detectedby immunostaining in 113 out of 147 breast carcinoma in both tumoral andstromal cells. It is expressed in both carcinoma and stromal cells in 88prostate carcinoma biopsies out of 98 (Ravel et al. 2008, Clin CancerRes 14, 1258-1265). TOP is responsible for the extracellular activationof succinyl-β-ALAL-doxorubicin and PhAc-ALGP-doxorubicin (Dubois et al.2006, Eur J Cancer 17, 3049-3056).

Dipeptidyl Prolyl Peptidases DPIV (CD26) and FAP

DPIV is a dipeptidylprolylpeptidase with a broad spectrum of activityand covers a large number of physiological substrates. It is expressedin epithelial cells of a large number of organs. It is expressed inthymus spleen and lymph nodes as well as lymphocytes. DPIV binds tocollagen and fibronectin in experimental conditions (Loster et al. 1995,Biochem Biophys Res Commun 217, 341). It is upregulated in the tumoralT-cell malignancies (Dang et al. 2002, Histol Histopathol 17, 1213-1226)and in different adenocarcinomas, such as in hepatocellular carcinoma(Stecca et al. 1997, J Hepatol 27, 997-945), thyroid carcinoma (Tanakaet al. 1995, Int J Cancer 64, 326-331), in meningiomas (Yu et al. 2010,FEBS Journal 277, 1126-1144; Stremenoova et al. 2010, Int J Oncology 36,351-358), in esophageal adenocarcinomas (Goscinski et al. 2008, APMIS116, 823-831), in lung adenocarcinomas (Asada et al. 1993,Histopathology 23, 265-270) and in bone and soft tissue tumors (Dohi etal. 2009, Histopathology 4, 432-440). DPIV is expressed in cancer stemcells of human colorectal cancer and of human mesotheliomas (Pang et al.2010, Cell Stem Cell 6, 603-615; Yamazaki et al. 2012, Biochem BiophysRes Commun 419, 529-536).

FAP is a dipeptidyl exopeptidase with very narrow specificity restrictedto glycine-proline, alanine-proline and lysine-proline dipeptides and isalso a type I collagenase. It can however also act as endopeptidase(Siew lai et al. 2007, Bioconj Chem 18, 1245-1250). FAP is absent innormal adult tissues such as epithelial, mesenchymal; neural andlymphoid cells such as lymphocytes. It is absent in non-malignanttumors. More importantly it is upregulated, not in the tumoral cellsthemselves, but in the reactive fibroblast, stromal and angiogenic cellspresent in epithelial and sarcoma tumors with the exception of the Ewingsarcoma (Yu et al. 2010, FEBS Journal 277, 1126-1144; Brennen et al.2012, Mol Cancer Ther 11, 257-269). It plays an important role in coloncancer (Leonard et al. 2007, Clin Cancer Res 13, 1736-1741), melanoma(Fazakas et al. 2011, PLoS one 6, e20758; Artym et al. 2002,Carcinogenesis 23, 1593-1601), pancreatic cancer (Hyung-Ok et al. 2011,BMC Cancer 11, 245; Min et al. 2012, World J Gastroenterol 28, 840-846),gastric cancer (Zhi et al. 2010, J Exp Clin Cancer Res 29, 66; Mori etal. 2004, Oncology 67, 411-419), non-small lung cancer (Bremnes et al.2011, J Thorac Oncol 1, 209-217), glioma (Menlein 2011, Biol Chem 392,199-207), skin cancers (Huber et al. 2006, J Cut Pathol 2, 145-155),cervical carcinoma (Jin et al. 2003, Anticancer Res 4, 195-0198),thyroid carcinoma (Nocolini et al. 2011, Biochem Pharmacol 7, 778-780),rectal carcinoma (Saaigusa et al. 2011, Int J Oncol 3, 655-663),esophageal carcinoma (Goscinski et al. 2008, Ultrastruct Pathol 3,89-96), in breast cancer (Huang et al. 2011, Clin Exp Metatstasis 6,567-579), and in bone and soft tissue tumors (Dohi et al. 2009,Histopathology 4, 432-440). The reactive stromal cells of tumors cellsare essential for the growth of the tumoral cells as well as for theirinvasive and angiogenic capacities (Santos et al. 2009, J Clin Invest119, 3613-3625; Cheng et al. 2002, Cancer Res 62, 4767-4772; Huang etal. 2004, Cancer Res 64, 2712-2716).

Based on the Examples (see, e.g. Example 16), several methods ofscreening for candidate prodrugs according to the invention can beenvisaged. Such methods include methods of screening candidate prodrugshaving the general structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a peptide with a minimum length of 4 consecutive amino        acids (tetrapeptide) and a maximum length of 8 amino acids (i.e.        a peptide with a length of 4, 5, 6, 7 or 8 consecutive amino        acids), which comprises carboxy-terminally a proline comprising        dipeptide selected from the group consisting of glycine-proline        (GP), alanine-proline (AP), and lysine-proline (KP);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacer group such as a        self-immolating or self-eliminating spacer group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group and/or are individually linked to D directly or        via a linker or spacing group; and        wherein said screening method is comprising the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to a GP-, AP- or KP-dipeptide to        obtain a GP-D, AP-D or KP-D as dipeptide-drug intermediate        prodrug;        (iii) contacting each of drug D and said dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D independently with in        vitro cultured cells;        (iv) determining the cytotoxicity of drug D and dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D;        (v) identifying from (iv) a dipeptide-drug intermediate prodrug        GP-D, AP-D or KP-D with comparable cytotoxic activity as drug D;        and        (vi) selecting [C_(x)-OP]_(y)-D corresponding to dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D identified in step (v)        as candidate prodrug.

In the above method, the term “corresponding to” is to be understoodsuch that the selected candidate prodrug [C_(x)-OP]_(y)-D is comprisingthe same drug D as present in the dipeptide-drug intermediate prodrugGP-D, AP-D or KP-D identified to have comparable cytotoxic activity asdrug D. Optionally, the drug D is connected to the oligopeptide moietyOP in the same was as it is connected to the GP-, AP- or KP-dipeptide inthe dipeptide-drug intermediate prodrug GP-D, AP-D or KP-D. In otherwords, the success of the in step (v) identified dipeptide-drugintermediate prodrug GP-D, AP-D or KP-D is indicative or predictive forthe success of the candidate prodrug [C_(x)-OP]_(y)-D wherein OPrepresents a peptide with a minimum length of 4 consecutive amino acids(tetrapeptide) and a maximum length of 8 amino acids (i.e. a peptidewith a length of 4, 5, 6, 7 or 8 consecutive amino acids) whichcomprises carboxy-terminally a proline comprising dipeptide selectedfrom the group consisting of glycine-proline (GP), alanine-proline (AP),and lysine-proline (KP). Such extrapolation is plausible in view of theextensive results described herein as obtained with doxorubicin as drugD. When a dipeptide-drug intermediate prodrug GP-D, AP-D or KP-D has acytotoxic activity comparable to the cytotoxic activity of drug D, thisis a good indication of the successful activation of such prodrug to thefree drug D by the cultured cells. The cultured cells used in this typeof screening may for instance be a cultured tumor cell line.

In a particular embodiment the peptide OP in the above mentioned generalstructure is a tetrapeptide with the sequence ALGP (SEQ ID NO: 1), ALAP(SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ IDNO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), orKLKP (SEQ ID NO: 10). Thus in said embodiment the present inventionprovides methods of screening candidate prodrugs having the generalstructure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a tetrapeptide with the sequence ALGP (SEQ ID NO: 1), ALAP        (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ        ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID        NO:9), or KLKP (SEQ ID NO:10); in particular OP is the        tetrapeptide ALGP (SEQ ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacer group such as a        self-immolating or self-eliminating spacer group, and wherein,        if y is greater than 1, the multiple OP moieties are        individually linked to each other directly or via a linker or        spacing group and/or are individually linked to D directly or        via a linker or spacing group; and        wherein said screening method is comprising the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to a GP-, AP- or KP-dipeptide to        obtain a GP-D, AP-D or KP-D as dipeptide-drug intermediate        prodrug;        (iii) contacting each of drug D and said dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D independently with in        vitro cultured cells;        (iv) determining the cytotoxicity of drug D and dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D;        (v) identifying from (iv) a dipeptide-drug intermediate prodrug        GP-D, AP-D or KP-D with comparable cytotoxic activity as drug D;        and        (vi) selecting [C_(x)-OP]_(y)-D corresponding to dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D identified in step (v)        as candidate prodrug.

Alternatively, said methods are methods of screening candidate prodrugshaving the general structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a tetrapeptide with the sequence ALGP (SEQ ID NO: 1), ALAP        (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ        ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID        NO:9), or KLKP (SEQ ID NO:10); in particular OP is the        tetrapeptide ALGP (SEQ ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group such as a        self-immolating or self-eliminating spacer, and wherein, if y is        greater than 1, the multiple OP moieties are individually linked        to each other directly or via a linker or spacing group and/or        are individually linked to D directly or via a linker or spacing        group;        wherein said method is comprising the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to [C_(x)-OP]_(y) to obtain a        [C_(x)-OP]_(y)-D prodrug;        (iii) contacting each of drug D and prodrug [C_(x)-OP]_(y)-D        independently with in vitro cultured cells;        (iv) determining the cytotoxicity of drug D and prodrug        [C_(x)-OP]_(y)-D;        (v) identifying from (iv) a prodrug [C_(x)-OP]_(y)-D with        comparable cytotoxic activity as drug D; and        (vi) selecting [C_(x)-OP]_(y)-D identified in step (v) as        candidate prodrug.

In the above mentioned screening methods, the term “comparable cytotoxicactivity” is to be understood such that a prodrug, after contact withthe in vitro cultured cells (such as cultured tumor cells), exerts atleast 50% or at least between 50 and 99% (e.g., at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least55%, at least 90%, at least 95%, at least 99%) of the cytotoxic activityexerted by the free drug contacted with the same in vitro cultured cellsunder the same conditions.

In another alternative, said methods are methods of screening candidateprodrugs having the general structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a tetrapeptide with the sequence ALGP (SEQ ID NO: 1), ALAP        (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ        ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID        NO:9), or KLKP (SEQ ID NO:10); in particular OP is the        tetrapeptide ALGP (SEQ ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group such as a        self-immolating or self-eliminating spacer, and wherein, if y is        greater than 1, the multiple OP moieties are individually linked        to each other directly or via a linker or spacing group and/or        are individually linked to D directly or via a linker or spacing        group;        wherein said method is comprising the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to [C_(x)-OP]_(y) to obtain a        [C_(x)-OP]_(y)-D prodrug;        (iii) contacting prodrug [C_(x)-OP]_(y)-D for 5 h at 37° C. with        in vitro cultured cells;        (iv) determining the conversion of prodrug [C_(x)-OP]_(y)-D into        free drug D;        (v) identifying from (iv) a prodrug [C_(x)-OP]_(y)-D which is        converted by at least 50% to D; and        (vi) selecting [C_(x)-OP]_(y)-D identified in step (v) as        candidate prodrug.

In yet another alternative, said methods are methods of screeningcandidate prodrugs having the general structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a tetrapeptide with the sequence ALGP (SEQ ID NO: 1), ALAP        (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ        ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID        NO:9), or KLKP (SEQ ID NO:10); in particular OP is the        tetrapeptide ALGP (SEQ ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group such as a        self-immolating or self-eliminating spacer, and wherein, if y is        greater than 1, the multiple OP moieties are individually linked        to each other directly or via a linker or spacing group and/or        are individually linked to D directly or via a linker or spacing        group;        wherein said method is comprising the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to a GP-, AP- or KP-dipeptide to        obtain a GP-D, AP-D or KP-D dipeptide-drug intermediate prodrug;        (iii) contacting dipeptide-drug intermediate prodrug GP-D for 3        h at 37° C. with isolated FAP and/or DPIV peptidases;        (iv) determining the conversion of dipeptide-drug intermediate        prodrug GP-D, AP-D or KP-D into free drug D;        (v) identifying from (iv) a dipeptide-drug intermediate prodrug        GP-D, AP-D or KP-D which is converted by at least 50% to D; and        (vi) selecting [C_(x)-OP]_(y)-D corresponding to dipeptide-drug        intermediate prodrug GP-D, AP-D or KP-D identified in step (v)        as candidate prodrug.

In the above method, the term “corresponding to” is to be understoodsuch that the selected candidate prodrug [C_(x)-OP]_(y)-D is comprisingthe same drug D as present in the prodrug GP-D identified to beconverted by at least 50% into D under the defined conditions.Optionally, the drug D is connected to the oligopeptide moiety OP in thesame was as it is connected to the GP-dipeptide in the prodrug GP-D. Inother words, the success of the in step (v) identified prodrug GP-D isindicative or predictive for the success of the candidate prodrug[C_(x)-OP]_(y)-D wherein OP is the ALGP-peptide. Such extrapolation isplausible in view of the extensive results described herein as obtainedwith doxorubicin as drug D.

Further envisaged methods include methods of screening candidateprodrugs having the general structure

[C_(x)-OP]_(y)-D,

wherein C is a capping group;

-   -   OP is a tetrapeptide with the sequence ALGP (SEQ ID NO: 1), ALAP        (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ        ID NO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID        NO:9), or KLKP (SEQ ID NO:10); in particular OP is the        tetrapeptide ALGP (SEQ ID NO: 1);    -   D is a drug;    -   x is an integer being at least 1 when y=1;    -   y is an integer being at least 1, if y is greater than 1, then        at least 1 OP is carrying a capping group; and        wherein the linkage between C and OP and the linkage between OP        and D is direct or via a linker or spacing group such as a        self-immolating or self-eliminating spacer, and wherein, if y is        greater than 1, the multiple OP moieties are individually linked        to each other directly or via a linker or spacing group and/or        are individually linked to D directly or via a linker or spacing        group;        wherein said method is comprising the steps of:        (i) obtaining the drug D;        (ii) conjugating the drug D to [C_(x)-OP]_(y) to obtain a        [C_(x)-OP]_(y)-D prodrug;        (iii) contacting prodrug [C_(x)-OP]_(y)-D for 3 h to 24 h at        37° C. with isolated CD10 and/or TOP peptidases and with        isolated FAP and/or DPIV peptidases;        (iv) determining the conversion of prodrug [C_(x)-OP]_(y)-D into        free drug D;        (v) identifying from (iv) a prodrug [C_(x)-OP]_(y)-D which is        converted by at least 50% to D; and        (vi) selecting [C_(x)-OP]_(y)-D identified in step (v) as        candidate prodrug.

In any of the above screening methods referring to conversion of aprodrug GP-D or of a prodrug [C_(x)-OP]_(y)-D to drug D, the conversionpercentage used for selecting the candidate prodrug in general lieswithin at least 50 to 100% (e.g. at least 50%, e.g., at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 55%, at least 90%, at least 95%, at least 99%).

In any of the above alternative screening methods said capping group Cmay be a phosphonoacetyl group. In a particular embodiment, the OP inany one of the above screening methods is a peptide with a minimumlength of 4 consecutive amino acids (tetrapeptide) and a maximum lengthof 8 amino acids (i.e. a peptide with a length of 4, 5, 6, 7 or 8consecutive amino acids) which comprises carboxy-terminally a prolinecomprising dipeptide selected from the group consisting ofglycine-proline (GP), alanine-proline (AP), and lysine-proline (KP);more in particular a tetrapeptide with the sequence ALGP (SEQ ID NO:1),ALAP (SEQ ID NO:3), TSGP (SEQ ID NO:4), TSAP (SEQ ID NO:5), KLGP (SEQ IDNO:6), KLAP (SEQ ID NO:7), ALKP (SEQ ID NO:8), TSKP (SEQ ID NO:9), orKLKP (SEQ ID NO: 10); even more in particular OP is the tetrapeptideALGP (SEQ ID NO: 1).

In any of the above alternative screening methods said drug D may beselected from the group consisting of doxorubicin, maytansine,geldanamycin, paclitaxel, docetaxel, campthothecin, vinblastine,vincristine, vindesine, methothrexate, aminopterin, amrubicin, or aderivative of any thereof.

The invention further relates to kits comprising a container comprisinga prodrug or salt thereof according to the invention or comprising acomposition comprising such prodrug or salt thereof. Such kit mayfurther comprise, in the same container (holding a prodrug or saltthereof according to the invention) or in one or more separatecontainers, one or more further anticancer drugs, such as an antibody orfragment thereof (e.g. as described above). Alternatively, or inaddition, such kit may further comprise, in the same container (holdinga prodrug or salt thereof according to the invention) or in one or moreseparate containers, one or more drug resistance reversing agents. Otheroptional components of such kit include one or more diagnostic agentscapable of determining the success of a therapy comprising a prodrug orsalt thereof according to the invention; use instructions; one or morecontainers with sterile pharmaceutically acceptable carriers, excipientsor diluents; one or more containers with agents for ADEPT therapy; etc.

All references hereinabove and hereinafter cited are incorporated intheir entirety by their reference.

EXAMPLES Example 1. Synthesis of N-Capped Peptide Prodrug Compounds 1.Synthesis of Fmoc-Peptide-OH 1.1. Synthesis of Fmoc-ALAL-OH

The Fmoc-Leu-Wang resin (5 g, 1 eq) was swollen in dimethylformamide (20mL) for 30 minutes. The Fluorenylmethyloxycarbonyl (Fmoc)-group wasremoved by treatment with piperidine (4 mL) in dimethylformamide (16 mL)for 3 minutes, the resin was then filtered, followed by the sametreatment for 3 and 7 minutes. The resin was washed withdimethylformamide (20 mL) three times. Fmoc-Ala-OH (2 eq) and2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (2 eq) were solubilised in dimethylformamide (20 mL)and N,N-diisopropylethylamine (4 eq) was added. The mixture waspreactivated during 6 minutes and added to the resin. The resin was thenshaken for 60 minutes and washed three times with dimethylformamide (20mL). The Fmoc group was removed by treatment with piperidine (4 mL) indimethylformamide (16 mL) for 3 minutes, the resin was then filtered.The same treatment was repeated twice for 3 and 7 minutes. The resin waswashed three times with dimethylformamide (20 mL). The same protocol wasrepeated with Fmoc-Leucine-OH (2 eq) and with Fmoc-Alanine-OH (2eq).

After the last coupling, the resin was washed alternatingly withdimethylformamide (20 mL) and Dichloromethane (20 mL) three times anddried. The Fmoc peptide was cleaved from the resin with a solution oftrifluoroaceticacid/triisopropylsilane/water (95:2.5:2.5 v/v/v) (20 mL)during 2 hours. The solvent was evaporated. The product was precipitatedin water and filtered. The Fmoc-peptide was dried by lyophilization.

MS (ES+): 609.3 [MH]⁺; Purity: 90% (determined by HPLC at 214 nm).

1.2. Synthesis of Fmoc-ALG-OH

Prepared as described in paragraph 1.1. starting with a Fmoc-Gly-Wangresin instead of Fmoc-Leu-Wang resin and adding Fmoc-Leu and Fmoc-Ala.

MS (ES⁺): 482.2 [MH]⁺; Purity: 92% (determined by HPLC at 214 nm).

1.3. Synthesis of Fmoc-ALPF-OH

Prepared as described in paragraph 1.1. starting with a Fmoc-Phe-Wangresin instead of Fmoc-Leu-Wang resin and adding Fmoc-Pro; Fmoc-Leu andFmoc-Ala.

MS (ES+): 669 [MH]+; Purity: 98% (determined by HPLC at 214 nm).

1.4. Synthesis of Fmoc-ALAF-OH

Prepared as described in paragraph 1.3. starting with a Fmoc-Phe-Wangresin and adding Fmoc-Ala; Fmoc-Leu and Fmoc-Ala.

MS (ES+): 643 [MH]+; Purity: 90% (determined by HPLC at 214 nm).

1.5. Synthesis of Fmoc-AIG-OH

Prepared as described in paragraph 1.1. starting with a Fmoc-Gly-Wangresin instead of Fmoc-Leu-Wang resin and adding Fmoc-Ile and Fmoc-Ala.

MS (ES⁺): 482.5 [MH]⁺; Purity: 60% (determined by HPLC at 214 nm).

1.6. Synthesis of Fmoc-KLG-OH

Prepared as described in paragraph 1.1. starting with a Fmoc-Gly-Wangresin instead of Fmoc-Leu-Wang resin and adding Fmoc-Leu and Fmoc-Lys(IvDde).

MS (ES⁺): 744 [MH]⁺

1.7. Synthesis of Fmoc-GPG-OH

Prepared as described in paragraph 1.1. starting with a Fmoc-Gly-Wangresin instead of Fmoc-Leu-Wang resin and adding Fmoc-Pro and Fmoc-Gly.

MS (ES⁺): 452 [MH]⁺

2. Synthesis of Peptide-Doxorubicin Conjugates 2.1. Synthesis ofNH₂-ALAL-Doxorubicin

Doxorubicin (1 eq) was solubilised in dimethylformamide (10 mL). Asolution of Fmoc-ALAL-OH (1.2 eq) in dimethylformamide (2 mL) was addedto the doxorubicin and the pH was adjusted to pH 8 withN,N-diisopropylethylamine. The solution was stirred at RT and the2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (1.2 eq) in dimethylformamide (2 mL) was added. ThepH of the solution was checked and readjusted to pH 8-8.5. The solutionwas stirred at room temperature and was checked by HPLC. If the reactionwas complete, the Fmoc group was removed by treatment with piperidine(10% final volume) during 5 minutes at RT and the lactate buffer 10% pH3 was added at 0° C. The mixture was loaded on YMC. The product wasrecovered with MeOH and the solvent was evaporated. The ALAL-Doxorubicinwas purified by HPLC semi-preparative (column Luna, C18).

MS (ES⁺): 912 [MH]⁺; Purity: 95% (determined by HPLC at 214 nm).

2.2. Synthesis of NH₂-Pro-Doxorubicin and NH₂-Gly-Pro-Doxorubicin

Doxorubicin (1 eq) was solubilised in dimethylformamide (10 mL). Asolution of Fmoc-Proline-OH (1.2 eq) in dimethylformamide (2 mL) wasadded to the doxorubicin and the pH was adjusted to pH 8-8.5 withN,N-diisopropylethylamine. The solution was stirred at RT and the2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (1.2 eq) in dimethylformamide (2 mL) was added. ThepH of the solution was checked and readjusted to pH 8-8.5. The solutionwas stirred at room temperature and was checked by HPLC. If the reactionwas complete, the Fmoc group was removed by treatment with piperidine(10% final volume) during 5 minutes at RT and the lactate buffer 10% pH3 was added at 0° C. The mixture was loaded on YMC. The product wasrecovered with MeOH and the solvent was evaporated. The Pro-Doxorubicinwas purified by HPLC semi-preparative (column Luna, C18). The sameprotocol was followed with the Fmoc-Glycine-OH (1.2 eq).

P-Dox: MS (ES⁺): 641 [MH]⁺; Purity: 99% (determined by HPLC at 214 nm).

GP-Dox: MS (ES⁺): 698 [MH]⁺; Purity: 99% (determined by HPLC at 214 nm).

2.3. Synthesis of NH₂-ALGP-Doxorubicin Conjugate

Pro-Doxorubicin (1 eq) was solubilised in dimethylformamide (10 mL). Asolution of Fmoc-ALG-OH (1.2 eq) in dimethylformamide (2 mL) was addedto the doxorubicin and the pH was adjusted to pH 8 withN,N-diisopropylethylamine. The solution was stirred at RT and the2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (1.2 eq) in dimethylformamide (2 mL) was added. ThepH of the solution was checked and readjusted to pH 8-8.5. The solutionwas stirred at room temperature and was checked by HPLC. If the reactionwas complete, the Fmoc group was removed by treatment with piperidine(10% final volume) during 5 minutes at RT and the lactate buffer 10% pH3 was added at 0° C. The mixture was loaded on YMC. The product wasrecovered with MeOH and the solvent was evaporated. The ALGP-Doxorubicinwas purified by HPLC semi-preparative (column Luna, C18).

MS (ES+): 882 [MH]+; Purity: 99% (determined by HPLC at 214 nm).

2.4. Synthesis of NH₂-ALPF-Doxorubicin

Prepared as described in 2.1 using Fmoc-ALPF-OH instead of Fmoc-ALAL-OH.

MS (ES+): 971[MH]+; Purity: 97% (determined by HPLC at 214 nm).

2.5. Synthesis of NH₂-ALAF-Doxorubicin

Prepared as described in 2.1 using Fmoc-ALAF-OH instead of Fmoc-ALAL-OH.

MS (ES+): 947[MH]+ Purity: 96% (determined by HPLC at 214 nm).

2.6. Synthesis of NH₂-AIGP-Doxorubicin

Prepared as described in 2.1 using Fmoc-AIGP-OH instead of Fmoc-ALAL-OH.

2.7. Synthesis of NH₂-GPGP-Doxorubicin

Prepared as described in 2.1 using Fmoc-GPGP-OH instead of Fmoc-ALAL-OH.

3. Synthesis of PhAc-Peptide-Doxorubicin 3.1. Synthesis ofPhAc-ALGP-Doxorubicin

NH₂-ALGP-Dox (1 eq) was solubilised in dimethylformamide (10 mL). Asolution of Phosphonoacetic acid (2.5 eq) in dimethylformamide (2 mL)was added to the peptide-doxorubicin and the pH was adjusted to pH 8with N,N-diisopropylethylamine. The solution was stirred at RT and the2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (2 eq) in dimethylformamide (2 mL) was added. The pHof the solution was checked and readjusted to pH 8-8.5. The solution wasstirred at room temperature and was checked by HPLC. If the reaction wascomplete, the mixture was precipitated with diethylether and filtered.The product was recovered with MeOH and the solvent was evaporated. ThePhAc-ALGP-Doxorubicin was purified by HPLC semi-preparative (columnLuna, C18).

MS (ES+): 1004.4 [MH ]+; Purity: 99% (determined by HPLC at 214 nm).

3.2. Synthesis of PhAc-ALAL-Doxorubicin

NH₂-ALAL-Dox (1 eq) was solubilised in dimethylformamide (10 mL). Asolution of Phosphonoacetic acid (2.5 eq) in dimethylformamide (2 mL)was added to the peptide-doxorubicin and the pH was adjusted to pH 8with N,N-diisopropylethylamine. The solution was stirred at RT and the2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (2 eq) in dimethylformamide (2 mL) was added. The pHof the solution was checked and readjusted to pH 8-8.5. The solution wasstirred at room temperature and was checked by HPLC. If the reaction wascomplete, the mixture was precipitated with diethylether and filtered.The product was recovered with MeOH and the solvent was evaporated. ThePhAc-ALAL-Doxorubicin was purified by HPLC semi-preparative (columnLuna, C18).

MS (ES+): 1034 [MH ]+; Purity: 99% (determined by HPLC at 214 nm).

3.3. Synthesis of PhAc-ALPF-Doxorubicin

Prepared as described in 3.2 with NH₂-ALPF-Dox instead of NH₂-ALAL-Dox.

MS (ES+): 1094 [MH ]+; Purity: 92% (determined by HPLC at 214 nm).

3.4. Synthesis of PhAc-ALAF-Doxorubicin

Prepared as described in 3.2 with NH₂-ALAF-Dox instead of NH₂-ALAL-Dox.

MS (ES+): 1068[MH ]+; Purity: 97% (determined by HPLC at 214 nm).

3.5. Synthesis of PhAc-DLGP-Doxorubicin

PhAc-D(Dmab)LGP-doxorubicin was prepared as described in 3.2 withNH₂-D(Dmab)LGP-Dox instead of NH₂-ALAL-Dox. The protecting group Dmab,also known as4-{N-[1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}benzyl, was removed with hydrazine hydrate 2% during 5 minutes at roomtemperature. The lactate buffer 10% pH 3 was added at 0° C. and themixture was loaded on YMC. The product was recovered with MeOH and thesolvent was evaporated. The PhAc-DLGP-Doxorubicin was purified by HPLCsemi-preparative (column Luna, C18).

MS (ES+): 1048.3 [MH ]+; Purity: 99% (determined by HPLC at 214 nm).

3.6. Synthesis of PhAc-TSGP-Doxorubicin

PhAc-T(Dmab)SGP-doxorubicin was prepared as described in 3.2 withNH₂-T(Dmab)SGP-Dox instead of NH₂-ALAL-Dox. The protecting group Dmabwas removed with hydrazine hydrate 2% during 5 minutes at roomtemperature. The lactate buffer 10% pH 3 was added at 0° C. and themixture was loaded on YMC. The product was recovered with MeOH and thesolvent was evaporated. The PhAc-TSGP-Doxorubicin was purified by HPLCsemi-preparative (column Luna, C18).

MS (ES): 1008.3 [MH ]+; Purity: 99% (determined by HPLC at 214 nm).

3.7. Synthesis of PhAc-AIGP-Doxorubicin

Prepared as described in 3.2 with NH₂-AIGP-Dox instead of NH₂-ALAL-Dox.

MS (ES+): 1004.3[MH ]+; Purity: 99% (determined by HPLC at 214 nm).

3.8. Synthesis of PhAc-KLGP-Doxorubicin

PhAc-K(IvDde)LGP-doxorubicin was prepared as described in 3.2 withNH₂-K(IvDde)LGP-Dox instead of NH₂-ALAL-Dox. The protecting group IvDdewas removed with hydrazine hydrate 2% during 5 minutes at roomtemperature. The lactate buffer 10% pH 3 was added at 0° C. and themixture was loaded on YMC. The product was recovered with MeOH and thesolvent was evaporated. The PhAc-KLGP-Doxorubicin was purified by HPLCsemi-preparative (column Luna, C18).

MS (ES+): 1061 [MH ]+; Purity: 91.5% (determined by HPLC at 214 nm).

3.9. Synthesis of PhAc-GPGP-Doxorubicin

Prepared as described in 3.2 with NH₂-GPGP-Dox instead of NH₂-ALAL-Dox.

MS (ES+): 974.9[MH ]+; Purity: 98% (determined by HPLC at 214 nm).

Similar synthesis procedures as described above were used for thesynthesis of PhAc-TSGP-doxorubicin and PhAc-KLGP-doxorubicin

4. Synthesis of succinyl-βALAL-doxorubicin and ofsuccinyl-βALPF-doxorubicin

Succinyl-βALPF-doxorubicin

Succinyl-βALAL-doxorubicin and Succinyl-βALPF-doxorubicin were generatedas previously described by Fernandez A M et al., J. Med. Chem.,44:3750-3753 (2001).

Example 2. In Vitro Evaluation of N-Capped Peptide Prodrugs Stability inBlood and Plasma Method

Citrated human blood and plasma from healthy donors (pH 7, InnovativeResearch) was used to assess the stability of drug conjugates cappedwith phosphonoacetyl (PhAc-ALAL-Dox, PhAc-ALPF-Dox, PhAc-ALAF-Dox,PhAc-ALGP-Dox, PhAc-DLGP-Dox and PhAc-KLGP-Dox) in comparison with theknown Succinyl-βALAL-Dox prodrug conjugate.

The drug conjugates (50 μM) were mixed with human plasma and incubatedfor 5 hours at 37° C. in a water bath. Fifty μL of samples werecollected after 0, 1, 3 or 5 hours and an extraction was performedimmediately: 150 μL of acetonitrile was added to the 50 μL samples.Samples were vortexed and centrifuged for 10 min at 13 000 rpm at roomtemperature. Supernatant was collected. Samples were buffered byaddition of 200 mM formiate buffer pH 4.5 (1 V sample supernatant+3 Vbuffer) before HPLC analysis (fluo detection ex=235 nm em=560 nm).

Results

Similarly to Suc-βALAL-Dox, all tested capped conjugates were shown tobe stable in human plasma and human blood. After 5 hours of incubationat 37° C. in the presence of blood or plasma, 10% or less of metabolicderivatives of the doxorubicin conjugates were detected in testedsamples (Exception: PhAc-ALAL-Dox giving 15% metabolites in blood; datasummarized in Table 1 and 2).

TABLE 1 % metabolites released after 5 hours incubation at 37° C. incitrated human blood total Compounds Dox L-dox AL-Dox GP-dox F-Dox P-Dox% metabolites Suc-βALAL-Dox 2 6 1 — — — 9 PhAc-ALAL-Dox 9 6 0 — — — 15PhAc-ALPF-Dox 4 — — — 1 — 5 PhAc-ALAF-Dox 5 — — — 5 — 10 PhAc-ALGP-Dox 6— — 3 — 0 9 PhAc-DLGP-Dox 2 — — 0 — 2 4 PhAc-KLGP-Dox 2 — — 1 — 0 8LGP-Dox 71 8 GP-Dox 71

TABLE 2 % metabolites released after 5 hours incubation at 37° C. incitrated human plasma (pH 7) total Compounds Dox L-dox AL-Dox GP-doxF-Dox P-Dox % metabolites Suc-βALAL-Dox <1 2 0 — — — 2 PhAc-ALAL-Dox <12 0 — — 2 2 PhAc-ALPF-Dox 0 — — — 0 — 0 PhAc-ALAF-Dox 0 — — — 2 — 2PhAc-ALGP-Dox 2 — — 0 — 0 2 PhAc-DLGP-Dox 2 — — 0 — 1 3 PhAc-GPGP-Dox 6— — 0 — 0 6 PhAc-TSGP-Dox 4 — — 4 — 0 8 LGP-Dox 63 2 GP-Dox 50

Example 3. In Vitro Evaluation of N-Capped Peptide Prodrugs EnzymaticActivation 1. Method

1.1. Reactivation assay in the presence of tumor cell secretedpeptidases Sub-confluent cultures of LS-174T tumor cells were washedtwice with a saline phosphate buffer solution, and fresh culture medium(DMEM-F12 without phenol red) containing 0.02% bovine serum albumin isadded (100 μl/cm²). After 24 hours incubation, the conditioned medium iscollected, centrifuged for 10 minutes at 300 g, buffered with 1 MTris-HCl, pH 7.4 (1 volume of buffer+19 volumes of medium) andconcentrated 20 times by ultracentrifugation (cutoff threshold of 10kDa).

Drug compounds (50 μM) were incubated for 0, 1, 3 or 5 hours at 37° C.in the presence of freshly prepared LS174T tumor cell conditionedmedium. Fifty μL of sample were collected at each time point andprocessed as described above for human plasma.

1.2. Reactivation Assay in the Presence of Purified Enzymes (TOP, CD10,CD26, FAP)

CD10 (recombinant human neprilysin, R&D systems, Ref. 1182-ZN) wasdiluted at 20 nM in 0.1M MES pH 6.5 supplemented with 0.2 mg/mL BSA. TOP(recombinant human thimet oligopeptidase, R&D systems, Ref. 3439-ZN) wasdiluted at 10 nM in a solution of 50 mM Tris-HCl pH 7.4/0.5M NaCl/0.1MDTT. CD26 and FAP were diluted at 1 μg/mL in a Tris-HCl pH 7.5 buffer.Reactions were initiated by addition of 50 μM of each compound toenzymes solutions (1 V enzyme solution+1 V 100 μM drug solution).Samples were incubated for 0, 1, or 3 hours at 37° C. in a water bath inthe presence of purified enzymes. Fifty μL of sample were collected ateach time point and processed as described above for human plasma.Activation of Suc-βALAL-dox was tested in parallel as a reference.

1.3. Results

Results of in vitro reactivation assays of N-capped-peptide-Doxorubicinconjugates are presented in Table 3.

Suc-βALAL-Dox is cleaved by CD10 to release 73% of L-Dox. On thecontrary, PhAc-ALAL-Dox is not efficiently cleaved by CD10. Less than 5%of L-Dox is released after 3 hours of incubation in the presence of theenzyme. In this example, replacement of the succinyl-βA- by thephosphonoacetyl capping group inhibits the peptide-enzyme interaction.PhAc-ALAF-Dox is moderately cleaved by CD10 into F-Dox. Changing theALAF peptide moiety into ALPF, inhibits cleavage by CD10 whatever thecapping group used. PhAc-ALGP-Dox is cleaved by the enzyme into LGP-Dox(25% metabolite released after 3 hours of incubation at 37° C.).PhAc-AIGP-Dox is cleaved by CD10 into IGP-Dox (18% hydrolysis after 3hours of incubation). PhAc-DLGP-Dox and PhAc-GPGP-Dox, PhAc-KLGP-Dox andPhAc-TSGP-Dox are not or very slightly activated by CD10.

TOP hydrolyses Suc-βALAL-Dox and PhAc-ALAL-Dox to release 64% and 44% ofAL-Dox respectively. None of the ALAF-Dox and ALPF-Dox derivatives arecleaved by TOP.

TOP activates PhAc-ALGP-Dox, PhAc-KLGP-Dox and PhAc-TSGP-Dox into GP-Dox(72, 31 and 38% of metabolite detected after 3 hours of incubation inthe presence of the enzyme). PhAc-DLGP-Dox, PhAc-GPGP-Dox PhAc-AIGP-Doxare not cleaved by TOP.

The sensitivity of the capped tetrapeptidic Dox prodrugs to TOP and CD10is not contradictory with their blood stability. Most of the bloodpeptidases are exoproteases for which the capped tetrapeptidic Doxprodrugs are inaccessible as substrates. TOP and CD10 are, however,endoproteases unaffected by the presence of the capping group.

Suc-βALAL-Dox, and PhAc-ALAL-Dox are activated by tumor cell secretedenzymes to release L-Dox and to a lesser extent AL-Dox and Dox.PhAc-ALAF-Dox, PhAc-ALPF-Dox, and Suc-βALPF-Dox are all activated intoF-Dox by tumor cell secreted enzymes. PhAc-ALGP-Dox is hydrolyzed in thepresence of tumor cell conditioned medium into GP-Dox and Dox (56% and24% of all metabolites detected after 5 hours, respectively), whereasmainly GP-Dox is detected after PhAc-KLGP-Dox and PhAc-TSGP-Doxactivation. PhAc-DLGP-Dox, PhAc-AIGP-Dox and PhAc-GPGP-Dox are notactivated in the presence of LS174T tumor cells secreted peptidases.

No significant differences between PhAc- and Suc-βA-protecting groupsare shown for the ALAL-Dox, and ALPF-Dox derivatives.

In these experiments PhAc-ALAL-Dox and PhAc-ALGP-Dox compounds arebetter cleaved by tumor cell secreted enzymes and have been selected forfurther in vivo analysis.

Neither CD26 nor FAP hydrolized either PhAc-ALGP-Dox, Suc-βALAL-Dox orLGP-Dox, in line with these enzymes beingexoproteases/dipeptidylproline-proteinases. GP-Dox, however, is a goodsubstrate for both CD26 (97% conversion to Dox after 3 h incubation at37° C.) and FAP (62% conversion to Dox after 3 h incubation at 37° C.).

Cultured tumor cells are able to convert PhAc-ALGP-Dox to GP-Dox,LGP-Dox to Dox, and GP-Dox to Dox, which indicates the presence of CD10and/or TOP (PhAc-ALGP-Dox to LGP-Dox or GP-Dox) at the one hand and ofCD26 and/or FAP (GP-Dox to Dox) at the other hand. This also confirms atwo-step activation process of the prodrug occurring fullyextracellularly, i.e., intracellular (lysosomal) processing of theprodrug is not required. This in contrast to L-Dox (released fromSuc-βALAL-Dox) that is internalized and further hydrolysedintracellularly into Dox.

TABLE 3 % % Metabolites Metabolites % Metabolites CD10 released afterTOP released LS174T released after Compounds 10 nM 3 h 5 nM after 3 hCM20xcc 5 h Suc-βALAL-Dox + 73 ± 17 + 64 ± 10 + 65 ± 17 L-Dox AL-DoxDox + AL-Dox + L-Dox Suc-βALPF-Dox −* <5 − 0 + 17 ± 2 F-Dox F-DoxPhAc-ALAL- −* <5 + 44 ± 6 + 65 ± 19 Dox L-Dox AL-Dox Dox + AL- Dox +L-Dox PhAc-ALPF-Dox − 0 −* <5 + 11 ± 6 F-Dox F-Dox PhAc-ALAF-Dox + 24 ±5 −* <5 + 32± 15 F-Dox + LAF- F-Dox F-Dox Dox PhAc-ALGP-Dox + 25 ± 4 +72 ± 14 + 81 ± 31 LGP-Dox GP-Dox Dox + GP- Dox + LGP- Dox PhAc-DLGP-Dox− 0 − 0 − 0 PhAc-AIGP-Dox + 18 ± 3 −* <5 −* 3 ± 3 IGP-Dox GP-Dox Dox +GP- Dox + IGP-Dox PhAc-KLGP-Dox −* <5 + 31 ± 5 + 39 ± 13 LGP-Dox GP-DoxDox + GP-Dox PhAc-GPGP-Dox − 0 − 0 − 0 PhAc-TSGP-Dox − 0 + 38 ± 2 + 35 ±18 GP-Dox Dox + GP-Dox LGP-Dox − − + Dox (23%) + GP-Dox (77%) GP-Dox Dox(28%) *less than 5% of metabolites detected

Subsequently, the sensitivity of GP-Dox (released from PhAc-ALGP-Dox byTOP) as substrate to the activity of prolyl peptidases was tested inmore detail two such enzymes, CD26 (synonym DPIV) and FAP, are emergingas potentially important factors in cancer chemotherapy. CD26 (1 μg/ml)or FAP did not hydrolyze either of PhAc-ALGP-Dox, LGP-Dox (efficientlyreleased from PhAc-ALGP-Dox by TOP) or Suc-βALAL-Dox. However, GP-Doxwas shown to be a good substrate for CD26 and FAP and was cleaved intoDox (97% conversion by CD26; 62% conversion by FAP). It is important tonotice that in this way, the specificity of PhAc-ALGP-Dox to cancercells is increased. TOP is involved in the prodrug activation process ofSuc-βALAL-Dox or PhAc-ALAL-Dox (releasing AL-Dox) and of PhAc-ALGP-Dox(releasing GP-Dox). While AL-Dox is more generally sensitive toconversion into L-Dox and Dox by cell-secreted peptidases (and withL-Dox being automatically hydrolyzed into Dox intracellularly), GP-Doxseems sensitive to peptidases predominantly released by tumor cells,such as CD26 and FAP. This difference in sensitivity and the differencein enzymes involved in the activation of GP-Dox into Dox (compared toactivation of AL-Dox into Dox) are likely to result in differences intoxicities and activities between e.g. PhAc-ALGP-Dox on the one hand andSuc-βALAL-Dox or PhAc-ALAL-Dox on the other hand. Based on this, and asdescribed in the next Example, the in vivo toxicity of PhAc-ALGP-Dox wasassessed in comparison with PhAc-ALAL-Dox.

Example 4. Evaluation of the In Vivo Toxicity of PhAc-ALGP-Dox andPhAc-ALAL-Dox Conjugates after Single or Multiple Intravenous Injectionsin Mice Method

PhAc-ALAL-Dox and PhAc-ALGP-Dox were dissolved in saline. Compounds wereadministered by intravenous bolus injection in the lateral tail vein ofOF-1 mice (10 μl/g). The in vivo toxicity was evaluated by monitoringbody weight.

Results

Results in FIG. 1 show the high toxicity of PhAc-ALAL-Dox at 160μmol/kg. No significant body weight loss is observed in the grouptreated with PhAc-ALAL-Dox at 80 μmol/kg. PhAc-ALGP-Dox injection at thedoses of 240 and 320 μmol/kg is well tolerated. A moderate body weightloss with a maximum of 15% at day 28 is recorded in the PhAc-ALGP-Dox240 μmol/kg treated group showing its lower toxicity in comparison withPhAc-ALAL-Dox. Injection of PhAc-ALGP-Dox at 320 μmol/kg induced asignificant body weight loss and one mouse was found dead at day 12.These data indicate that the maximum tolerated dose (MTD) ofPhAc-ALGP-Dox after a single iv bolus injection is between 240 and 320μmol/kg. The toxicity of Dox varies between 30 and 40 μmol/kg indicatingthat after one intravenous injection PhAc-ALGP-Dox is at least 6 timesless toxic.

Example 5. Effectiveness Studies of PhAc-ALGP-Dox after Repeated ivBolus Injections in Human Xenograft Tumor Models in Nude Mice Method

The anti-tumor activity of doxorubicin and PhAc-ALGP-Dox was tested inmodels of athymic mice (nude/nude NMRI) carrying ectopic xenografts ofhuman LS-174T colon carcinoma or MX-1 mammary carcinoma.

LS 174T and MX-1 tumors were established by a subcutaneous implantationof cells (3×10⁶ and 10⁷ cells injected respectively) in the right flankof 6 weeks old female NMRI nude mice (Harlan). Treatments were initiatedwhen the tumors had reached a size of 150-200 mm³ (calculated using thefollowing formula: [length×width²]/2). The day of the first injectionanimals were randomly assigned to groups of 4 animals. Doxorubicin, andPhAc-ALGP-Dox were dissolved in saline. Compounds were delivered bybolus intravenous injection (i.v.) in the lateral tail vein at 10 μl/g.During the course of the experiment, clinical signs, body weight andtumor volume were controlled twice a week. Results are presented as theevolution of mean tumor volume as a function of time. Optimal T/C (ratioof mean tumor volume of treated versus control mice) values were used asa measure of treatment efficacy. The optimal T/C % reflects the maximaltumor growth inhibition achieved (TGI=100-(T/C′ 100)).

Results

As shown in FIG. 2, PhAc-ALGP-Dox was injected twice (once weekly) i.v.at 140 mole/kg and 160 mole/kg in nude mice bearing subcutaneouslyimplanted LS174T tumors (colon carcinoma). Their body weight, and thetumor size were followed for 28 days and compared with the Dox (15μmol/kg) and NaCl treated animals groups. Significant and similarantitumor activity was observed in all treated groups. (Table 4).

These data also confirm the lower toxicity of PhAc-ALGP-Dox since at thedoses of 140 μmol/kg and 160 μmol/kg the body weight loss (maximum 10%)was comparable to that induced by Dox given at a 9 times lower dose.

TABLE 4 Tumor Growth Inhibition. Mean RTV and standard deviation werecalculated for each group at the end of the study. Drug efficacy wasexpressed as the percentage tumor growth inhibition (% TGI), calculatedusing the equation 100 − (T/C′ 100), where T is the mean RTV of thetreated tumor and C is the mean RTV in the control group. compoundPhAc-ALGP-Dox PhAc-ALGP-Dox Doxorubicin Dose 140 μmol/kg 160 μmol/kg 15μmol/kg TGI [% (day)] 79 (28) 81 (28) 55 (28)

In another experiment, PhAc-ALGP-Dox was injected four times i.v. (atday 0, 3, 6 and 9) at the dose of 100 μmol/kg in nude mice bearingsubcutaneously implanted MX-1 tumors (mammary carcinoma). Their bodyweight and the tumor size were followed for 29 days and compared withthe Dox (8 μmol/kg) and NaCl treated animals groups. No significant bodyweight loss and similar significant antitumor activity (inhibition oftumor growth>60%) were observed for the 2 tested drugs (FIG. 3 and Table5).

TABLE 5 Tumor Growth Inhibition. Mean RTV was calculated for each groupat the end of the study. Drug efficacy was expressed as the percentagetumor growth inhibition (% TGI), calculated using the equation 100 −(T/C′ 100), where T is the mean RTV of the treated tumor and C is themean RTV in the control group. compound PhAc-ALGP-Dox Doxorubicin Dose100 μmol/kg 8 μmol/kg TGI [% (day)] 60 (29) 65 (29)

Example 6. Pharmacokinetic and Tissue Quantification of PhAc-ALGP-Doxand its Metabolites after Single iv Bolus Injection in Mice inComparison with Doxorubicin at Equimolar Dose Method

Pharmacokinetic tissue distribution studies were performed using OF-1mice. Doxorubicin and PhAc-ALGP-Dox were dissolved in saline at the doseof 8.62 mM and administered to mice by the i.v. route in the lateraltail vein (10 μL/g). At different time points after drug administration(5 min, 30 min, 1 h, 4 h, 7 h, 16 h and 24 h) 3 mice per group weresacrificed by cervical dislocation and blood and heart tissue werecollected. Hearts were incised and rinsed carefully in phosphate buffersaline (to eliminate blood in the cardiac cavities), dried on paper andfrozen in liquid nitrogen. They were stored until analysis. Bloodsamples were centrifuged (10 min, 2000 g, 4° C.) to separate the plasmafraction, which was stored for analysis. The hearts were homogenizedwith an Ultraturrax homogenizer in 1.5 mL water. The proteinconcentration was measured using the microBCA protein assay (Pierce).The drug tissue quantification was made by HPLC after extraction: 150 μLof acetonitrile was added to the 50 μL samples. Samples were vortexedand centrifuged for 10 min at 13 000 rpm at room temperature.Supernatant was collected. Samples were buffered by addition of 200 mMformiate buffer pH 4.5 (1 V sample supernatant+3 V buffer) before HPLCanalysis (fluo detection ex=235 nm em=560 nm).

Results

Doxorubicin and PhAc-ALGP-dox were injected i.v. bolus at the equimolardose of 86.2 mol/kg to wild type female OF-1 mice. Evolution of drug andmetabolites concentration in plasma and cardiac tissue was determined byHPLC analysis. About 90% of the drug plasma concentration was eliminatedin the first five minutes after injection of Dox or PhAc-ALGP-Dox (FIGS.4 A and B). Less than 1% of the PhAc-ALGP-Dox was rapidly hydrolysedinto LGP-Dox, GP-Dox and Dox. These metabolites were no longer detectedafter 1 hour. The plasma area under curve (AUC) value for Doxorubicinafter injection of Doxorubicin is 63 times higher than after injectionof PhAc-ALGP-Dox (Table 6).

Since the heart is the target for an important toxicity of Doxorubicin,the cardiac tissue concentration of the free drug was determined. Alsodetermined were the heart AUCs for Doxorubicin after injection ofDoxorubicin, and heart AUCs for Dox, GP-Dox and PhAc-ALGP-Dox afteradministration of PhAc-ALGP-Dox (Table 6). The Dox heart AUC afterPhAc-ALGP-Dox administration is 25 times lower than after Doxadministration at equimolar dose. Given the clinical cardiotoxic effectof the Dox AUC, these results strongly suggest that PhAc-ALGP-Dox wouldbe significantly less cardiotoxic than Doxorubicin.

TABLE 6 Pharmacokinetic AUC values of PhAc-ALGP-Dox and of itsmetabolites vs Doxorubicin in cardiac tissue after one i.v. bolusinjection to OF-1 mice at the dose of 86.2 μmol/kg AUC PhAc-ALGP-Dox(area under curve) Dox Dox GP-Dox PhAc-ALGP-Dox Plasma (μM · h ) 63 1 197 Heart (pmol · h/mg protein) 5863 235 11 23

Example 7. In Vitro Cytotoxicity Assay of PhAc-ALGP-Dox onCardiomyocytes

The in vitro cardiotoxicity test was carried out in a relevant andpredictive in vitro model for cardiac safety screening in early leadoptimization using mouse embryonic stem cell derived cardiomyocytes(Cor.At®, Axiogenesis (Germany)). Cor.At® cardiomyocytes provide astandardized, homogenous and reproducible cell system for the in vitroclassification of a compound's cardio-cytotoxic potential. Afterincubation with test compounds, the neutral red uptake test was used todetermine effects which directly affect the viability and integrity ofcardiac cells when compared to a non-specific reference cell type, e.g.mouse fibroblasts (MEF).

Results

CorAt cardiomyocytes were incubated in the presence of increasingconcentrations of PhAc-ALGP-Dox or of Doxorubicin. Cell viability wasdetermined after 48 h using the neutral red uptake test. Mouse embryonicfibroblasts (MEF) were used as control cells to distinguish cardiacspecific toxicity from general cytotoxicity. The dose response curve ofPhAc-ALGP-Dox (FIG. 5) did show a moderate toxic effect on Cor.Atcardiomyocytes only at the highest concentration tested (20 μg/ml). Atthis concentration, the effect on MEF is less pronounced (81% viabilityvs. 37% viability). For the MEF, no IC50 is reached with this compound.At all lower concentrations tested PhAc-ALGP-Dox did not show any toxiceffect. The dose response curve of Doxorubicin did show a severe toxiceffect on Cor.At cardiomyocytes as well as on MEF at the two highestconcentrations tested (20 μg/ml and 2 μg/ml). At 0.2 μg/ml, the compounddid show a moderate toxic effect on Cor.At cardiomyocytes, but only amarginal effect on MEF (67% viability of Cor.At cardiomyocytes vs. 89%viability of MEF). Although the effect on Cor.At cardiomyocytes is onlyslightly higher than on MEF, the compound is considered to exert acardiotoxic effect, which may be masked by a general cytotoxic effect.

As illustrated in FIG. 5, this study shows that PhAc-ALGP-Dox is 40 to50 times less cytotoxic than Dox on Cor.At® cardiomyocytes.

Example 8. Assessment of PhAc-ALGP-Dox Activation at the Tumor Siteafter Single iv Bolus Injection in Nude Mice Bearing LoVo ColonCarcinoma Xenograft Method

The tumor activation of PhAc-ALGP-Dox was assessed using athymic mice(nude/nude NMRI) carrying ectopic xenograft of human LoVo coloncarcinoma. LoVo tumors were established by a subcutaneous implantationof cells (10⁷ cells) in the right flank of 6 weeks old female NMRI nudemice (Harlan). Drugs or controls were administered four weeks aftersubcutaneous implantation of the xenograft. On the day of injection,animals were randomly assigned to groups of 4 animals. The PhAc-ALGP-Doxconjugate was dissolved in saline at increasing doses (1.5, 3.5, 5, 10,20, 30, 46, and 62 mM). The conjugates were delivered by bolusintravenous injection (i.v.) in the lateral tail vein at 10 μl/g.Twenty-four hours after injection, mice were sacrificed by cervicaldislocation and tumors were collected, rinsed in phosphate buffer salineand homogenized. An extraction of drugs from tumor homogenates wasperformed with acetonitrile and Doxorubicin present in tumors wasquantified by HPLC analysis.

Results

Results in FIG. 6 show that Dox tumor concentration increases with theinjected dose of PhAc-ALGP-Dox to reach a plateau value at 200 μmol/kg.Results of this example indicate that a limited prodrug activation rateand availability at the tumor site could depend on the maximum of enzymeactivity available during the duration of the contact withPhAc-ALGP-Dox.

Example 9. Evaluation of the In Vivo Toxicity of PhAc-ALGP-Dox afterSingle and Multiple Intraperitoneal Injections in Mice Method

PhAc-ALGP-Dox was dissolved in saline and administered by single ormultiple intraperitoneal (ip) injections in the lateral tail vein ofOF-1 mice (10 μl/g). PhAc-ALGP-Dox was administered at similarcumulative doses of 280 and 560 μmol/kg following different injectionsschedules: single ip injection; 5 consecutive daily ip injections at 56and 112 μmol/kg or twice a day for five consecutive days at the doses of28 and 56 μmol/kg. The in vivo toxicity was evaluated by monitoring thebody weight.

Results

Whatever the injection schedule, no body weight loss was recorded inanimal groups having received the cumulative dose of 280 μmol/kg ofPhAc-ALGP-Dox (FIG. 7). The toxicity study of PhAc-ALGP-Dox after singleip injection at 280 μmol/kg was made separately and results are notshown in FIG. 7. The dose of 560 μmol/kg administered by single ipinjection was very toxic. Animals lost 25% of body weight in one weekand were sacrificed. Results clearly show that fractionation of the dosein multiple injections reduces the toxicity. Five consecutive daily ipinjections of PhAc-ALGP-Dox at 112 μmol/kg also induced a significantbody weight loss with a maximum of 22.5% at day 11 but followed by arecovery phase. No body weight loss was observed in the group treatedwith 10 ip injections (twice a day for five consecutive days) ofPhAc-ALGP-Dox at 56 μmol/kg. Considering that the maximum tolerated doseof doxorubicin injected according the same regime is 3 μmol/kg,PhAc-ALGP-doxorubicin is, in these conditions, about 15 times lesstoxic.

Example 10. Pharmacokinetic and Tissue Quantification of PhAc-ALGP-Doxand its Metabolites after Single Intraperitoneal Injection in Mice inComparison with Doxorubicin at Equimolar Dose Method

Pharmacokinetic tissue distribution studies were performed using OF-1mice. Doxorubicin and PhAc-ALGP-Dox were dissolved in saline at the doseof 9.2 mM and administered to mice by intraperitoneal route (10 μL/g, 6mice per group). At different time points after drug administration (5min, 30 min, 1 h, 4 h, and 24 h), blood samples were collected from thelateral tail vein of three mice using EDTA-coated microtubes (Starsted).After 24 h, 3 mice per group were sacrificed by cervical dislocation andhearts were collected. They were incised and rinsed carefully inphosphate buffer saline (to eliminate blood in the cardiac cavities),dried on paper and frozen in liquid nitrogen. They were stored untilanalysis. The hearts were homogenized with an Ultraturrax homogenizer in1.5 mL water. The protein concentration was measured using the microBCAprotein assay (Pierce). The drug tissue quantification was made by HPLCafter extraction: 150 μL of acetonitrile was added to the 50 μL samples.Samples were vortexed and centrifuged for 10 min at 13 000 rpm at roomtemperature. Supernatant was collected. Samples were buffered byaddition of 200 mM formiate buffer pH 4.5 (1 V sample supernatant+3 Vbuffer) before HPLC analysis (fluo detection ex=235 nm em=560 nm).

Results

The pharmacokinetics of PhAc-ALGP-Dox in blood was evaluated afterintraperitoneal (ip) injection to OF-1 mice. A low percentage (about 1%)of the injected dose reached the blood compartment in the first fiveminutes after ip injection of PhAc-ALGP-Dox at 92 μmol/kg. The bloodconcentration of the prodrug was stable for one hour and subsequentlydecreased. The conjugate was no longer detected after 4 hours (FIG. 8A).The AUC values were 44.2 μM.h and 3.6 μM.h for PhAc-ALGP-Dox and Doxrespectively. Results in FIG. 8B show the pharmacokinetics in blood ofDoxorubicin at equimolar dose. A low percentage (about 2.5%) of theinjected dose reached the blood compartment in the first five minutesafter injection. The blood concentration of Dox decreased rapidly withinone hour to a very low concentration that remained stable up to 24 hoursafter injection. The AUC value for Doxorubicin was 70.3 μM.h.

The Doxorubicin cardiac tissue concentration was measured 24 h after ipinjection of PhAc-ALGP-Dox or Doxorubicin at equimolar dose. Results inTable 7 show that Doxorubicin accumulates 19 times less afterintraperitoneal injection of 92 μmol/kg PhAc-ALGP-Dox than afterinjection of Doxorubicin at equimolar dose.

TABLE 7 In vivo cardiac concentration of Dox after intraperitonealinjection of Dox and PhAc-ALGP-Dox at 92 μmol/kg. Mice were sacrificed24 hours after drug administration and hearts were collected. Drugconcentration was determined by HPLC analysis after extraction fromtissue homogenates. Results are expressed in pmol/mg protein ± SD(concentrations of drugs and proteins were corrected taking into accountof the blood remaining in the cardiac tissue). Dox concentration inheart after 24 h treatments pmol/mg protein Dox 92 μmol/kg 253 ± 60PhAc-ALGP-Dox 92 μmol/kg 13 ± 3

Example 11. Evaluation of the In Vivo Efficacy of PhAc-ALGP-Dox afterRepeated Intraperitoneal Injections in Human Xenograft Tumor Models inNude Mice Method

The efficacy of PhAc-ALGP-Dox was assessed using athymic mice (nude/nudeNMRI) carrying ectopic xenograft of human LoVo colon carcinoma or ofMX-1 mammary carcinoma in comparison with free Doxorubicin. Tumors wereestablished by a subcutaneous implantation of cells (10⁷ cells) in theright flank of 6 weeks old female NMRI nude mice (Harlan). Treatmentswere administered when the tumors reached a size of 150-200 mm³(measured using a caliper and calculated with the following formula:[length×width²]/2). Animals were randomly assigned to groups of 4 to-6animals. Doxorubicin and PhAc-ALGP-Dox were dissolved in saline.Compounds were delivered by bolus intraperitoneal injection (ip) at 10μl/g. During the course of the experiment, clinical signs, body weightand tumor volume were controlled twice a week. Results are presented asthe evolution of mean tumor volume as a function of time. Optimal T/C(ratio of mean tumor volume of treated versus control mice) values andTGD (tumor growth delay in reaching 1000 mm³) were used as a measure oftreatment efficacy. The optimal T/C % reflects the maximal tumor growthinhibition (TGI) achieved (TGI=100−(T/C′100)). A statistical analysiswas performed at day 22 using the Mann Whitney t test of the Graph PadPrism 5.0 software.

Results

Mice bearing Lovo xenografts received twice a day (at 5-6 h interval)for 5 consecutive days (2Q1D5; total of 10 injections) intraperitonealinjections of saline, or of Doxorubicin at 0.5, 1 and 2 μmol/kg, or ofPhac-ALGP-Dox at 25, 35 and 50 μmol/kg. Their body weight and the tumorsize were followed and compared (FIG. 9). Tumor measurements werestopped in NaCl and Doxorubicin treated groups when tumor necrosisoccurred. No significant body weight loss was recorded in thisexperiment. PhAc-ALGP-Dox induced a dose-dependent antitumor efficacyand increase in tumor growth delay (Table 8). A moderate tumor growthinhibition was observed in the group treated with Doxorubicin at 2μmol/kg whereas no antitumor activity was seen at the doses of 0.5 and 1μmol/kg. At day 22, antitumor efficacy was statistically higher with 50μmol/kg PhAc-ALGP-Dox when compared with 2 μmol/kg Dox with TGI valuesof 65% and 45% respectively. The absolute growth delay induced by eachtreatment was calculated as the time in days for tumors in treated miceto grow from 190 to 1,000 mm³ minus the time in days for tumors to reachthe same size in vehicle-treated mice. The 50 μmol/kg PhAc-ALGP-Doxtreatment protocol resulted in a growth delay of 17 days whereas thehighest dose of 2 μmol/kg Doxorubicin alone induced a growth delay ofonly 6 days.

TABLE 8 Tumor Growth Inhibition and Tumor Growth Delay. Drug efficacy isexpressed as the percentage tumor growth inhibition (% TGI), calculatedusing the equation 100-(T/C′100), where T is the mean Relative TumorVolume (RTV) of the treated tumor and C is the mean RTV as the in thecontrol group. The absolute growth delay induced by each treatment iscalculated time in days for tumors in treated mice to grow from 190 to1,000 mm³ minus the time in day for tumors to reach the same size invehicle-treated mice (TGD). compound PhAc-ALGP-Dox Doxorubicin Dose(μmol/kg) 25 35 50 0.5 1 2 TGI [% (day)] 32 (22) 60 (22) 65 (22) 5 (22)no 45 (22) TGD [days]  5 13 17 1   0 6

In another similar study (FIG. 10), drugs or controls were administeredfor 2 consecutive weeks (2Q1D5×2W; total of 20 injections). In thiscase, results clearly show the better efficacy and lower toxicity ofPhAc-ALGP-Dox compared to Doxorubicin. No significant body weight losswas observed in the PhAc-ALGP-Dox treated groups (max 13% at day 9 inthe group 50 mol/kg PhAc-ALGP-Dox). However a dose-dependent toxicitywas observed in the Doxorubicin treated groups. The dose of 1.5 and 2μmol/kg were very toxic and induced severe body weight loss and animaldeath. The dose of 1 μmol/kg was slightly less toxic but above the MTDsince a continuous loss of weight was observed (max 15% at day 29) andone dead mouse was found at day 29. At this dose, Doxorubicin had a verylow activity with a TGI value of 44% at day 29. The highest efficacy wasobtained with 50 μmol/kg PhAc-ALGP-Dox with a TGI value of 73% at day 29and a significant TGD of 22 days (Table 9).

TABLE 9 Tumor Growth Inhibition and Tumor Growth Delay. Drug efficacy isexpressed as the percentage tumor growth inhibition (% TGI), calculatedusing the equation 100-(T/C′100), where T is the mean RTV of the treatedtumor and C is the mean RTV in the control group. The absolute growthdelay induced by each treatment was calculated as the time in days fortumors in treated mice to grow from 190 to 1,000 mm³ minus the time indays for tumors to reach the same size in vehicle-treated mice (TGD).compound PhAc-ALGP-Dox Doxorubicin Dose 25 35 50 1 1.5 2 (μmol/kg) TGI[% (day)] 54 (29), n = 5 53 (29), n= 5 73 (29), n = 5 44 (29), n = 4 65(29), n = 1 — TGD [days]  9  9 22 5 9   —

MX-1 xenografted mice received at 72 h interval 2 cycles of 1 week with2 daily ip injections of PhAc-ALGP-Dox at 50 μmol/kg or of Doxorubicinat 1 μmol/kg or 1.5 μmol/kg (2Q1D5×2W; total of 20 injections).Doxorubicin at 1.5 μmol/Kg was very toxic and induced severe body weightloss and death of animals. No significant body weight loss was recordedfor Doxorubicin at 1 μmol/kg (MTD) and for 50 μmol/kg PhAc-ALGP-Dox.Results in FIG. 11 show that 50 mol/kg PhAc-ALGP-Dox significantlyinhibits tumor growth and has improved efficacy as compared with 1μmol/kg Doxorubicin (MTD). At the end of the study (day 35), percentagesof tumor growth inhibition were 76% and 44% for PhAc-ALGP-Dox or forDoxorubicin respectively (Table 10). In this study tumor necrosis wasobserved in all groups (mice with necrosed tumors were removed from thestudy).

These experiments confirmed the 25- to 50-times reduced toxicity ofPhAc-ALGP-Dox compared to doxorubicin.

TABLE 10 Tumor Growth Inhibition and Tumor Growth Delay. Drug efficacyis expressed as the percentage tumor growth inhibition (% TGI),calculated using the equation 100 − (T/C′ 100), where T is the mean RTVof the treated tumor and C is the mean RTV in the control group.compound PhAc-ALGP-Dox Doxorubicin Dose (μmol/kg) 50 1 1.5 TGI [% (day)]76 (35), n = 24 44 (35), n = 3 Not determined

Example 12. Evaluation of PhAc-ALGP-Dox Efficacy after RepeatedIntraperitoneal Injections in the B16F10 Melanoma Lung Metastasis Modelin Mice Method

The efficacy of PhAc-ALGP-Dox was tested in the well described B16-F10lung metastatic melanoma model. For that purpose, 5×10⁷ B16-F10 murinemelanoma cells were injected in the lateral vein of C57BL6 mice.Treatments started three days after cells injection. Animal receivedtwice a day (at 5-6 h interval) for 5 consecutive days intraperitonealinjections (total 10 injections/mouse) of saline or of Doxorubicin at 2and 3.5 μmol/kg or of PhAc-ALGP-Dox at 50 μmol/kg. Five mice per groupwere sacrificed at day 14 after cells injection. Lungs were collectedand processed for melanin quantification (Molecular Pharmacology, 74:1576-1586, 2008). Survival was determined by observation of theremaining mice.

Results

PhAc-ALGP-Dox significantly inhibits the formation of lung metastasisand increases survival of mice as compared with NaCl and Doxorubicintreated groups (FIG. 12). At day 14 the amount of melanin in lunghomogenate was 501 mg/mL in the control group, 183 and 53 mg/mL in the 2and 3.5 μmol/kg Doxorubicin treated groups and 16 mg/mL in the 50μmol/kg PhAc-ALGP-Dox treated group. The median survival was 20, 24, 28and 36 days in groups receiving NaCl, 2 and 3.5 μmol/kg Doxorubicin or50 μmol/kg PhAc-ALGP-Dox, respectively.

Example 13. Evaluation of the In Vivo Efficacy of PhAc-ALGP-Dox afterRepeated Intraperitoneal Injections in the Orthotopic HCT116 ColonCarcinoma Tumor Model in Mice Method

HCT116 cells were subcutaneously injected into SCID mice. Oncexenografts were established, they were excised and orthotopicallyimplanted into the ceacum of other female γ-irradiated SCID mice usingmicrosurgical techniques. On day 12 after the cancer cells injection,the mice were randomized in four groups of 16. They received for 5consecutive days 2 daily intraperitoneal injections of saline, ofDoxorubicin at 2 μmol/kg and of PhAc-ALGP-Dox at 35 and 50 μmol/kgrespectively. On day 34 after injection into the caecum of coloniccancer cells the animals were sacrificed, the number of metastases wascounted macroscopically and the primary tumors weighted.

Results

Results are depicted in FIGS. 15 and 16. Doxorubicin at 2 μmol/kg provedto be too toxic for SCID mice and all animals died within 10 days.PhAc-ALGP-Dox was also more toxic on these mice compared to the othertested animals tumor xenograft models and 12 mice survived 34 days atthe dose of 35 μmol/kg and 9 mice survived in the 50 μmol/kg treatedgroup.

The control group gave a mean primary tumor weight of 0.88 g with a SDof 0.41. The second group at 30 μmol/kg of PhAc-ALGP-Dox had a primarytumor weight of 0.69 g with a SD of 0.25 while the group at 50 μmol/kgPhAc-ALGP-Dox presented a significant tumor weight loss with 0.44 g witha SD of 0.10.

The number of hepatic metastases were respectively of 20 (SD of 33) andof 24 (SD of 26) for the controls and the group treated at 30 μmol/kgPhAc-ALGP-Dox. The effect of 50 μmol/kg PhAc-ALGP-Dox was verysignificant with a mean number of metastases of 1.78 with a SD of 2.9.

Although PhAc-ALGP-Dox was not totally devoid of toxicity, this prodrugcould at least be retained for treating/preventing metastases (e.g.hepatic metastases of colon carcinoma) if it would not be effectiveagainst the primary tumor itself (e.g. colon carcinoma) at non-toxiclevels.

Example 14. Evaluation of PhAc-ALGP-Dox Leucopenia in Comparison withDoxorubicin

The leucopenic effects of PhAc-ALGP-Dox (35 μmol/kg ip) and ofDoxorubicin (3.5 μmol/kg ip) were compared in two independentexperiments. CD1 mice received two daily intraperitoneal injections ofdrugs for five consecutive days (total 10 injections/mouse; 2×5 animalsper group). The mice body weight evolution was recorded. Blood wascollected from the tail vein in EDTA-coated Microvettes tubes (Starsted)at day 4, 11 and 15 after treatment initiation. White blood cells (WBC)were counted using the SCILvet abc hematologic analyzer. The increase ordecrease in WBC was expressed as a percentage of WBC on day 0 (100%) foreach mouse. FIG. 13 shows combined results of the two studies.

FIG. 13 A gives the mean and SD of the body weight variation of the twogroups and 13 B the white blood cells variations as a percentage of WBCon day 0 for each mouse. These results clearly indicate the absence oftoxicity of 35 μmol/kg PhAc-ALGP-Dox ip as compared to Doxorubicinadministered at a 10 times lower dose. No leucopenic effect and bodyweight loss were observed in the PhAc-ALGP-Dox treated group. On theother hand, in the doxorubicin treated group, one mouse was found deadat day 11 as well as 3 mice at day 15. Doxorubicin induced a moderate tosevere leukopenia (on average—43% WBC at day 15) and body weight loss(on average—15% at day 15).

Example 15. Evaluation of PhAc-ALGP-Dox Chronic Cardiotoxicity Method

Chronic cardiac toxicity of PhAc-ALGP-Dox in mice was morphologicallyevaluated as previously described by Bertazzoli et al. 1979 (CancerTreat Rep 63, 1877-1883). CD1 female white mice were treated by bolusintravenous injection in the tail vein at 10 μL/g (6 mice per group).Compounds were injected twice a week, ten times. Animals were nottreated for 2 weeks between the first four injections and the last sixinjections to allow recovery of the bone marrow depression. Thetreatment dose-levels of PhAc-ALGP-Dox were: 13.8; 27.6; 55.2; and 82.8mol/kg. Doxorubicin 6.9 μmol/kg was used as a reference. Three weeksafter the last injection, animals were deeply anaesthetized byintraperitoneal injection of nembutal (50 mg/kg) and exsanguinated. Thehearts were carefully collected, rinsed in NaCl 9‰ and fixed in a 10%formaldehyde solution. Samples were processed for histopathologicalanalysis (CITox Lab, France). The heart was trimmed, embedded inparaffin wax, sectioned at a thickness of 4 microns and stained withhematoxylin-eosin before microscopic evaluation. During the course ofthe experiment, body weight was controlled before each injection or oncea week.

Results

No body weight loss was observed in the groups treated withPhAc-ALGP-Dox (FIG. 14). At the end of the treatment, doxorubicintreated animals showed signs of weakness and decreased locomotoractivity. A moderate decrease in body weight was recorded in theDoxorubicin treated group with a maximum of 8% at the time of sacrifice.

Results of microscopic evaluation of cardiotoxicity are shown in Table11. One mouse of the Doxorubicin treated group was not submitted formicroscopic examination because of premature death (at day 17). In allmice given 6.9 μmol/kg Doxorubicin, there were microscopic cardiacchanges. There was minimal or slight vacuolation of the myocardiumcharacterized by the presence of small clear cytoplasmic vacuoles inmyofibers scattered in the ventricles, septum and atria. The nuclei ofmyofibers in the ventricles and septum were enlarged in 3 mice out of 5.In addition, there were atrophy and or lesions in the atrial myocardium,particularly on the left side. Atrial myofiber atrophy and inflammatoryinfiltrate were seen in 4 mice out of 5, along with fibrin thrombi in 3mice out of 5. In one of these mice, there was alsodegeneration/necrosis of myofibers. The administration of PhAc-ALGP-Doxdid not induce any pathologic microscopic findings in the heart at anydose levels (i.e. up to a 12 times higher dose as compared withDoxorubicin).

TABLE 11 Microscopic evaluation of PhAc-ALGP-Dox chronic cardiotoxicityin mice in comparison with Doxorubicin Treatment NaCl DoxorubicinPhAc-ALGP-Dox Dose (μmol/kg) 0 6.9 13.8 27.6 55.2 82.8 Number of mice 65 6 6 6 6 Vacuolation; myofib. 0 5 0 0 0 0 Grade 1 0 4 0 0 0 0 Grade 2 01 0 0 0 0 Thrombus; atrium 0 3 0 0 0 0 Grade 1 0 3 0 0 0 0 Atrophy;myofiber 0 4 0 0 0 0 Grade 1 0 1 0 0 0 0 Grade 2 0 3 0 0 0 0Degeneration/necrosis; 0 1 0 0 0 0 myocardium Grade 1 0 1 0 0 0 0Infiltration; mix. Cells 0 4 0 0 0 0 Grade 1 0 4 0 0 0 0 Enlarg. Nuclei;myofib. 0 3 0 0 0 0 Grade 1 0 3 0 0 0 0

General conclusion: the maximal tolerated dose of PhAc-ALGP-Dox is,depending on different schedules and mode of administration between 6 to16 times less toxic than Dox and significantly more active on threeexperimental tumor models (LS174T and MX-1 xenografts and the lungmetastasis B16 melanoma model). PhAc-ALGP-Dox does not induce leukopeniaat active doses and does not present cumulative cardiotoxicity in amouse model at a dose equivalent to 12 times that of Doxorubicin at itsMTD level.

Example 16. Synthesis and Evaluation of Other Cytotoxic CompoundsConjugated to PhAc-ALGP 1. PhAc-ALGP-Maytansine

Maytansine is a potent microtubule-targeted compound that inducesmitotic arrest and kills tumor cells at sub-nanomolar concentrations.However, its side effects and lack of tumor specificity have preventedsuccessful clinical use. It inhibits microtubule assembly, inducingmicrotubule disassembly, and disrupts mitosis. Maytansine exhibitscytotoxicity against many tumor cell lines and displays about 100-foldhigher cytotoxicity than the Vinca alkaloids and about 1000-fold highertoxicity than doxorubicin.

In clinical trials gastrointestinal and central neurologic toxicity weredose limiting whereas myelosuppression was infrequent. When evaluated asa single agent, maytansine failed to show any significant response inpatients with different types of cancers.

The figure below depicts maytansine:

Maytansine can be conjugated to PhAc-ALGP via a self-immolating spacerreacting with its free —NH or OH group. Maytansine is commerciallyavailable (e.g. Medkoo Biosciences; Xuzkou Kaiyide Chemical Co).

2. PhAc-ALGP-Geldanamycin

Geldanamycin and derivates thereof are a family of a benzoquinoneansamycins, antibiotics originally isolated on the basis of their weakantibiotic activity that were subsequently shown to display potentantitumor activity. Geldanamycin induces, compared to their normalcellular counterparts, preferential degradation of proteins that aremutated in tumor cells such as v-src, bcr-abl and p53. This effect ismediated via Hsp90. Despite its potent antitumor potential, geldanamycinhas several major disadvantages as antitumor agent which has led to thedevelopment of geldanamycin analogues, in particular analoguescontaining a substitution on the 17 position.

Derivatization of geldanamycin at the position leads to 17AAG(17-(allylamino)-17-demethoxygeldanamycin) hat has lower in vivotoxicity than geldanamycin. Even though Hsp90 affinity to 17AAG is lessthan to geldanamycin, 17AAG and geldanamycin gave biologic effects inmalignant cells at similar or same concentrations. Geldanamycin bindswith high affinity to the ATP binding pocket of Hsp90. Hsp90 is aubiquitous molecular chaperone critical for the folding, assembly andactivity of multiple mutated and overexpressed signaling proteins thatpromote the growth and/or survival of tumor cells. Binding ofgeldanamycin to Hsp90 causes the destabilization and degradation of itstarget. Burke et al. 2009 (Bioorg Med Chem Lett 19, 2650-2653) describeda technique to link geladanamycin to antibodies with a linker cleavableby lysosomal enzymes. This linker incorporates a (self-immolating)valine-alanine-p-aminobenzyl-amino moiety to allow attachment with theamino-group of geldanamycin on one hand and with a free amino-group ofthe antibody on the other hand. The same linking technique can be usedto obtain PhAc-ALGP-geldanamycin but after substitution in the linker ofthe valine-alanine dipeptide by the alanine-leucine-glycine-prolinetetrapeptide. Geldanamycin is commercially available (e.g. Calbiochem,Fermentec Biosciences, AG Scientific-Paclitaxel).

3. PhAc-ALGP-Paclitaxel and PhAc-Docetaxel

Taxanes are diterpenes produced by the Taxus plants. They includepaclitaxel (Taxol) and docetaxel (taxotere; see Figure below)

Paclitaxel is one of several cytoskeletal drugs that target tubulin.Paclitaxel-treated cells have defects in mitotic spindle assembly,chromosome segregation, and cell division. Paclitaxel stabilizes themicrotubule polymer, protecting it from depolymerisation, and therebyblocks mitosis. Recent studies have demonstrated that suppression ofdynamics occurs at concentrations lower than those needed to blockmitosis. At the higher therapeutic concentrations, paclitaxel appears tosuppress microtubule detachment from centrosomes, a process normallyactivated during mitosis. Paclitaxel is approved for treatment ofovarian, breast and lung cancers and Kaposi's sarcoma. Common sideeffects include nausea and vomiting, loss of appetite and haematologicaltoxicity such as neutropenia, anemia and thrombocytopenia, although someside effects are associated with the excipient used, Cremophor EL, apolyoxyethylated castor oil.

Docetaxel or taxotere differs from paclitaxel at two positions in itschemical structure. It has a hydroxyl functional group on carbon 10(where paclitaxel has an acetate ester), and a tert-butyl carbamateester exists on the phenylpropionate side chain instead of thebenzylamide in paclitaxel. The carbon 10 functional group change causesdocetaxel to be more water soluble than paclitaxel. The hydroxyl groupon carbon 2 remains unmodified.

Paclitaxel was linked to antibodies by a simple reaction (Guillemard &Saragovi 2001; Cancer Res 61, 694-699). Paclitaxel was derivatized byreacting glutaric aldehyde to give 2′-glutaryl-paclitaxel containing acleavable ester bond. 2′-glutaryl-paclitaxel was then activated byremoval of a hydroxyl group with carbodiimide and bound to an antibodydirectly via its amino-group to form a peptide linkage. This techniquewas used recently (Garcia et al Oncogene 2012; doi:10.1038/onc.2012.283) to link taxol to an antiherceptin monoclonalantibody. Experimental results indicate that the conjugate is active inexperimental tumors indicating that the drug is released in vivo.

Paclitaxel was also conjugated to antibodies after succinylation ofpaclitaxel at the 2′position and coupling to antibodies via an amidebound (Safavy et al. 2003; Bioconj Chem 14, 302-310). Similar methods ofconjugating activated 2′-glutaryl or 2′-succinyl paclitaxel to thePhAc-ALGP-tetrapeptide; alternatively a self-immolating spacer linked onthe carboxyl group of the succinyl-paclitaxel is used. And it isreasonable to expect release in vivo of taxol from thePhAc-ALGP-paclitaxel conjugate, certainly taking into account that atetrapeptide exerts a much smaller steric hindrance than antibodies do.Stability of the succinyl-linked prodrug conjugate in blood may be aproblem given the ester nature of the succinyl group on paclitaxel.Therefore it may be preferable to use a self-immolating group betweenthe 2′ carbon and the tetrapeptide. These conjugates methods could alsobe applied to docetaxel in view of unchanged 2′ carbon. Paclitaxel iscommercially available (e.g. Hulang Pharmaceutical, TradeIndia).

4. PhAc-ALGP-Camptothecin

Camptothecin (CPT; structure depicted below) is a cytotoxic quinolinealkaloid that inhibits the DNA enzyme topoisomerase I (Topol). CPTshowed remarkable anticancer activity in preliminary clinical trials butsuffers from low solubility and (high) adverse drug reaction. Because ofthese disadvantages synthetic and medicinal chemists have developednumerous syntheses of camptothecin and various derivatives. Two CPTanalogues have been approved and are used in cancer chemotherapy today:topotecan and irinotecan.

Studies have shown that substitution at position 7, 9, 10 and 11 canhave positive effect on CPT activity and physical properties, e.g.potency and metabolic stability. Enlargement of the lactone ring by onemethylene unit also enhances its abilities, as in homocamptothecin.Substitution at position 12 and 14 leads to an inactive derivative.

Burke et al. 2009 (Bioconjug Chem 20, 1242-1250) described the designand the synthesis of conjugates between antibodies and camptothecinanalogues. 7-butyl-10-aminocampthothecin and7-butyl-9-amino-10,11-methylenedioxy-camptothecine are 10- to 1000-timesmore potent than campthothecin and can be linked to antibodies via adipeptide linker with a selfimmolative spacer releasing the drugs inpresence of lysosomal enzymes. A similar technique is feasible to arriveat a conjugate of camptothecin or a derivative thereof with PhAc-ALGP.Camptothecin is commercially available (e.g. Calbiochem, SeebooDhakhwa).

5. PhAc-ALGP-vinblastine and PhAc-ALGP-vincristine

Vinblastine (structure depicted below) is an anti-microtubule drug usedto treat certain kinds of cancer, including Hodgkin's lymphoma,non-small cell lung cancer, breast cancer, head and neck cancer, andtesticular cancer. It is also used to treat Langerhans cellhistiocytosis. Vinblastine was traditionally obtained from Catharanthusroseus, also known as Vinca rosea, a Madagascar periwinkle. It isgenerated in the plant by the joining of the alkaloids catharanthine andvindoline.

At very low concentrations it suppresses microtubule dynamics and athigher concentrations it reduces microtubule polymer mass. Common sideeffects are low blood count of white and red blood cells, and plateletsmay temporarily decrease.

Vincristine is a close analog differing from vinblastine only by CHOinstead of CH3 on N₁. Although very similar to vinblastine in structureit has other therapeutic indications and a very severe side effect. Itsmain indications are non-Hodgkin's lymphoma, in acute lymphoblasticleukemia, and in treatment for nephroblastoma (Wilms' tumor, a kidneytumor most common in young children). The main side-effects ofvincristine are peripheral neuropathy, hyponatremia, constipation, andhair loss. Peripheral neuropathy can be severe, and be a reason toavoid, reduce, or stop the use of vincristine.

PhAc-ALGP-vinblastine or -vincristine conjugates can be obtained bylinking a desacetyl vinblastine or -vincristine via a self-immolativespacer bound on their carbon C4.

Kandukuri et al. 1985 (J Med Chem 28, 1079-1088) developed a synthesismethod of amino acid derivatives of vinblastine involving an amidelinkage with the carboxylic end side chain of the amino acid. Thelinkage was obtained by a mixed anhydride condensation between the C₄deacetylvinblastine and N-maleoyl amino acids; vinblastine-C4 amino acidmaleoyls were also conjugated to lactosaminated serum albumin and shownto be active against HepG2 carcinoma (Rao et al. 1989; Anticancer Res 9,973-979). Logically the same procedure is applicable to vincristine.Conjugation of vinblastine or vincristine to PhAc-ALGP is likewiseachievable with this method. The latter conjugates have a better safetyprofile than unconjugated vinblastine or vincristine while retaining theanticancer activity. Vinblastine is commercially available (e.g. MedkooBiosciences), as well as vincristine (e.g. Tocris Bioscience, MedkooBiosciences).

6. PhAc-ALGP-Methotrexate and PhAc-ALGPAminopterin

Methotrexate, is an antimetabolite and antifolate drug. It is used intreatment of cancer and of autoimmune diseases.

The similarity in structure of dihydrofolic acid (top) and methotrexate(bottom) suggests that methotrexate is a competitive inhibitor ofdihydrofolic acid.

Methotrexate was originally developed and continues to be used forchemotherapy either alone or in combination with other agents. It iseffective for the treatment of a number of cancers including: breast,head and neck, leukemia, lymphoma, lung, osteosarcoma, bladder, andtrophoblastic neoplasms. The most common adverse effects include:ulcerative stomatitis, low white blood cell count and thuspredisposition to infection, nausea, abdominal pain, fatigue, fever,dizziness, and acute pneumonitis.

Methotrexate is thought to affect cancer and rheumatoid arthritis by twodifferent pathways. For cancer, methotrexate allosterically inhibitsdihydrofolate reductase (DHFR), an enzyme that participates in thetetrahydrofolate synthesis. The affinity of methotrexate for DHFR isabout one 1000-fold that of folate. DHFR catalyses the conversion ofdihydrofolate to the active tetrahydrofolate. Methotrexate, therefore,inhibits the synthesis of DNA, RNA, thymidylates, and proteins. For thetreatment of rheumatoid arthritis, inhibition of DHFR is not thought tobe the main mechanism, but rather the inhibition of enzymes involved inpurine metabolism, leading to accumulation of adenosine, or theinhibition of T cell activation and suppression of intercellularadhesion molecule expression by T cells.

Umemoto et al. 1989 (Int J Cancer 43, 677-684) described a method tolink methotrexate via free carboxyl group to antibodies with a ofAla-Leu-Ala-Leu linker This method can likewise be applied to linkmethotrexate to PhAcALGP peptide and will restore the free carboxylgroup after enzymatic cleavage. Derivatives of the alpha-carboxylategroup are relatively non-active and non-toxic in vitro since a freealpha-carboxylate group is necessary for the binding of methotrexate toDHFR Potential prodrugs of methotrexate were also produced in which the2-aminogroup was acylated with alpha-amino acids (Smal et al. 1995;Biochemical Pharmacology 49, 567-574). These aminoacyl derivatives aresubstituted at the 2-NH₂ pteridine ring of methotrexate. Importantly,the 2-leucyl-methotrexate derivative is rapidly cleaved and activated inpresence of serum illustrating its sensitivity to serum exoproteases.This makes it plausible for a PhAc-ALGP-methotrexate conjugate todisplay tumor cell specific anticancer activity. Linking methotrexate toPhAc-ALGP is performed via the above-described technique. Methotrexateis commercially available (e.g. CF Pharma Ltd, Yaskika Pharmaceuticals).

Aminopterin (4-aminopteroic acid), a 4-amino analog of folic acid, is anantineoplastic drug with immunosuppressive properties. Aminopterin is asynthetic derivative of pterin. Aminopterin works as an enzyme inhibitorby competing for the folate binding site of the enzyme dihydrofolatereductase. Its structure is very similar to that of methotrexate and ithas also a 2-NH₂ on its pteridine moiety. Developed before methotrexate,it was superseded by the latter early in the 1950's because of itsgreater toxicity that could result from a greater activity. Greatereffectiveness was confirmed recently in the treatment of acute leukemia(Cole et al. 2005; Clin Cancer Res 11, 8089-8096). APhAc-ALGP-aminopterin conjugate can be synthesized by the methodsdescribed for PhAc-ALGP-methotrexate and enhanced specificity of theanticancer activity for tumor cells is likewise plausible. Aminopterinis commercially available (e.g. Cameo Chemicals, Sigma Aldrich).

7. PhAc-ALGP-Amrubicin

Amrubicin (structure depicted below) is a third-generation, syntheticanthracycline analogue that has demonstrated substantial clinicalefficacy in the treatment of small cell lung cancer. Amrubicin is apotent topoisomerase II inhibitor and is being studied as a single agentand in combination with anti-cancer therapies for a variety of solidtumors, including lung and breast. It has been granted the orphan drugclassification by the FDA.

It is an anthracycline that is structurally different from that ofdoxorubicin. However it possesses an NH₂ group on its tetracycline ring.Side effects are similar to that of doxorubicin such as neutropenia andthrombocytopenia. Nothing is known about chronic cardiotoxicity aspossible side-effect.

As outlined for doxorubicin, conjugation of PhAc-ALGP to thisanthracycline is applied. The presence of the PhAc-ALGP increases tumorcell selectivity of the anticancer activity of amrubicin. Amrubicin iscommercially available (e.g. Medkoo Biosciences, Santa Cruz Biotech).

8. Common Steps for the In Vitro and In Vivo Testing ofPhAc-ALGP-Cytotoxic Compound Conjugates

The synthesis of the derivatives will be based on the methods describedsummarily hereinabove and more detailed in the referenced publicationsmentioned for each cytotoxic compound.

In a first step, a GP-dipeptide is conjugated to the cytotoxic compoundand analytical methodology is developed for detecting and/or quantifyingthe GP-cytotoxic compound conjugate, the cytotoxic compound, andintermediates between the two. Such methodology may include one or moreof spectrophotometry, high performance liquid chromatography (HPLC),mass spectrometry (MS), combined HPLC and MS, NMR and MALDI-TOF(matrix-assisted laser desorption/ionization—Time-of-flight), or evenUPLC-MS/MS (ultra-high performance liquid chromatography with tandemmass-spectrometry).

The purified GP-cytotoxic compound conjugate is tested in vitro in abiological system to confirm its cytotoxicity. For GP-cytotoxic compoundconjugates confirmed to be cytotoxic, synthesis of the completePhAc-ALGP-cytotoxic compound conjugate is performed and analyticalmethodology is developed for detecting and/or quantifying thePhAc-ALGP-cytoyoxic compound conjugate, the cytotoxic compound, andintermediates between the two.

Although chemical synthesis and analytical method development areexpected to be routine, it may be desired to study possiblemodifications in the initially envisaged synthesis method or to developof a new synthesis method such as to e.g. increase yield and/or purityof the peptide-drug conjugate.

9. In Vitro Testing Steps 9.1. Biological In Vitro Testing of theGP-Cytotoxic Compound Conjugate

The cytotoxic activity of the starting compound (unconjugated, the“parent” drug) and of its conjugated derivative (the “GP-drug”) will betested on in vitro cell lines. The cell lines will be at least some ofthose mentioned in the referenced publications mentioned hereinabove foreach of the cytotoxic compounds. It will be necessary to do tests as afunction of drug concentration and time of incubation. The GP-drugshould be much less active than the parent drug after short incubationtimes. With increasing incubation time, this difference could becomeless significant due to increased hydrolysis of the GP-drug byexoproteases present in serum that is part of the incubation media. Insuch case, the presence of exoproteases in serum could be confirmed, ifpossible, by incubation of the GP-drug in serum-free incubation media.

More crucial, however, is the analysis of cytotoxicity of the GP-drugbefore and after preincubation with purified FAP and/or DPIVprolyl-peptidases. In analogy with GP-doxorubicin, action of FAP and/orDPIV prolyl-peptidases on the GP-drug should significantly increase thecytotoxicity through release of the free drug. If the results of thisanalysis are positive, synthesis of the PhAc-ALGP-drug is performed. Ifthe results are negative, this could result from the inaccessibility tothe enzymes of the proline-drug bond. A solution to this problem couldbe to intercalate a spacer with an available NH₂-terminal betweenproline and the drug provided that such derivative retains its originalcytotoxic effect. Another possible solution would be to intercalate aself-immolating spacer that restores the original drug after hydrolysisof the drug-spacer and proline bound. One possible spacer of this typeis PABC or PAB (para-aminobenzyloxycarbonyl), attaching the drug moietyto the ligand in the conjugate. The linker moiety comprises a peptidesequence that is a substrate for an extracellular enzyme, for exampleFAP, that cleaves the peptide at an amide bond. The peptide furthercontains a self-immolating moiety which connects the drug and theprotein peptide sequence. Upon cleavage of the peptide sequence by anintracellular enzyme the self-immolating moiety cleaves itself from thedrug moiety such that the drug moiety is in an underivatized and activeform.

9.2. Biological In Vitro Testing of the PhAc-ALGP-Cytotoxic Conjugate

When a satisfactory GP-prodrug is obtained, biological in vitrocharacteristics of the corresponding the PhAc-ALGP-cytotoxic compoundconjugate are analyzed. This analysis includes assessing the in vitrocell cytotoxic effects of the original, unconjugated, parent drug and ofits conjugated prodrug counterpart. Dose-response curves for drug andprodrug are compared. The prodrug is expected to exert comparablecytotoxicitcy as the unconjugated drug.

An alternative experiment consists of incubating the PhAc-ALGP prodrugwith purified CD10 and TOP, and analyze conversion to GP-prodrug; aftersimultaneous incubation of the PhAc-ALGP prodrug with CD10, TOP, FAP andDPIV, the extent of conversion of PhAc-ALGP prodrug to free drug can beanalyzed. Significantly high levels of conversion to GP-prodrug and freedrug, respectively, are indicative of cytotoxic efficacy of thePhAc-ALGP prodrug in a cellular/tumor environment.

Both above described methodologies can also be combined: thecytotoxicity to in vitro cultured cells of the reaction product ofPhAc-ALGP prodrug with purified CD10 and TOP, or of PhAc-ALGP prodrugwith CD10, TOP, FAP and DPIV, or of both, can be compared to that of thefree drug.

9.3. In Vitro Pharmacodynamics of GP-Cytotoxic Compound Conjugates andPhAc-ALGP-Cytotoxic Compound Conjugates

The intracellular uptake (rate) of a prodrug before and afterpreincubation with proteases as described above is studied. In order toachieve this, adequate labeling of drug and prodrug may be required (seefurther).

10. In Vivo Testing Steps On The PhAc-ALGP-Cytotoxic Compound Conjugate10.1. Determination of the Maximum Tolerated Dose (MTD)

In vivo testing first determines the MTD of the prodrug conjugate bymeasuring the weight loss of mice injected with increasing doses of theprodrug. This is compared with the MTD of the free cytotoxic drug. TheMTD will be determined as the dose not inducing a weight loss exceeding20% of the original weight of the animals.

Initially, the prodrug and the free drug will be administered IV 2 timeswith a weekly interval, possibly repeated one or more times. Based onthe herein described experience with the doxorubicin prodrug, in vivoactivation of the prodrug could be very (s)low. In such case, the MTD isdetermined in normal mice and in mice xenografted with a human tumor,after twice daily IP injections for 2 times 5 days. Alternatively slowinfusion of the prodrugs is possible using osmotic or other programmableminipumps.

The effect of the prodrugs on the blood white cells count will becompared with the free drug.

10.2. Chemotherapeutic Activity on Immunodeficient Mice Grafted withHuman Tumors

One or two different human xenograft tumor models are selected on thebasis of published data obtained with the unconjugated/free cytotoxiccompound. Prodrug and free drug are injected IP at the MTD determined asdescribed above. Body weight and tumor volume are measured every twodays and compared with results obtained with the free starting drug atits MTD.

This can be repeated on other human tumor types such as human leukemiason orthotopic human xenografted tumors in SCID mice. Besides follow-upof the cytotoxic effect of the prodrug and drug on the primary tumor,the effect on metastases can be determined.

10.3. Pharmacokinetics, Tumor- and Tissue Distribution ofPhAc-ALGP-Cytotoxic Compound Conjugates

Except perhaps for a fluorescent drug such as amrubicin, pharmacokinetictissue distribution studies of drug and prodrug may require adequatelabeling of drug and prodrug too yield sufficient sensitivity inchemical analytical determination methods. One type of labeling isradiolabeling, the compounds could e.g. be radiolabeled by tritiumexchange or by neosynthesis with C₋₁₄ labeled precursors. Labelingshould be such that the label, or a label is maintained in metabolitesof the prodrug. Plasma pharmacokinetics are explored as well as tumoraccumulation of the conjugated drug and its metabolites, combined withorgan and tissue distribution.

Example 17. In Vivo Efficacy of PhAc-ALGP-Doxorubicin in UZLX-STS3 SoftTissue Sarcoma Xenograft Model

From the results of the experiment presented in FIGS. 16 to 19, severalinteresting properties of PhAc-ALGP-dox can be derived.

Tumor Volume.

In this experiment the effect of linking doxorubicin to the ALGP peptidehas been assessed by testing its activity in doxorubicin resistantliposarcoma. The present results demonstrate that linking of doxorubicinto ALGP results in a higher tolerated experimental dose. As such, evenin the experimental chemotherapy of a doxorubicin resistant sarcomaxenograft, PhAc-ALGP-dox is capable to limit xenograft growth of saiddoxorubicin resistant liposarcoma. In this study groups of mice (n=4 inboth groups), with each mice bearing two tumors were respectivelytreated with saline (control group), treated with doxorubicin(doxorubicin-treated group) and treated with PhAc-ALGP-dox(PhAc-ALGP-dox-treated group) Between day 0 and day 21 of treatment inboth control (n=7) and doxorubicin-treated tumors (n=8) there was asteady increase of tumor volume to 258% (p=0.018) and 246% (p=0.012),respectively. On the other hand tumors treated with PhAc-ALGP-doxrevealed stabilization of tumor volume at 105% (FIG. 16). The delay intumor volume growth in PhAc-ALGP-dox treated mice (n=6) wasstatistically significant on day 21 when compared with the control group(p=0.003) or with the doxorubicin-treated group (p=0.002).

The observed effect of PhAc-ALGP-dox may in part be explained by the 7-dcontinuous administration of the drug via a minipump.

A second and more fascinating explanation resides in a probably muchhigher release of doxorubicin in the tumor stroma than afteradministration of free doxorubicin (which can only be administered at amuch lower dose). The cytostatic and cytotoxic effect of the releaseddoxorubicin on stromal cells may in turn strongly affect the growth ofthe cancerous cells. This stromal effect may also explain thestabilizing effect on the tumor volume for 21 days without a reductionin their volume. Extended observation beyond 21 days, possibly incombination with repeated administration, using for example differentvehicles with effect on active drug release, could synergisticallyresult in a reduced tumor volume (after the destruction of all tumoralstromal cells).

Body Weight.

During the whole experiment, mice body weight and general well-beingwere monitored. A detailed graph depicting body weight evolution ispresented in FIG. 17 (control group: n=4; PhAc-ALGP-dox-treated group:n=3; doxorubicin-treated group: n=4). No major side effects wereobserved and in general the animals' body weight did not drop below theacceptable value (20% of the starting body weight loss during thetreatment). One mouse was sacrificed for ethical reasons on day 13 inthe PhAc-ALGP-dox group (body weight 79.8%, animal getting skinny fromday 11, limited intraperitoneal ascites fluid found during the necropsyprobably due to infection as consequence of the surgical implantation ofthe minipump).

Total White Blood Cell Count and Total Neutrophil Count

No major changes in total white blood cells and neutrophils wereobserved in mice treated with PhAc-ALGP-dox in comparison with controlor doxorubicin-treated animals (FIGS. 18 and 19, respectively; controlgroup: n=4; PhAc-ALGP-dox-treated group: n=3; doxorubicin-treated group:n=4). Neutropenia is one of the most important toxic side effects offree doxorubicin-treatment. Therefore, unchanged netrophil count 21 daysafter administration of PhAc-ALGP-doxorubicin at 40-times higher dosethan free doxorubicin is from a clinical point of view a very promising,novel experimental result.

1. A prodrug having the general structure:[C_(x)-OP]_(y)-D, wherein C is a capping group; OP is an oligopeptidicmoiety; D is a drug; x is an integer being at least 1 when y=1; y is aninteger being at least 1, if y is greater than 1, then at least 1 OP iscarrying a capping group; and wherein the linkage between C and OP andthe linkage between OP and D is direct or via a linker or spacing group,and wherein, if y is greater than 1, the multiple OP moieties areindividually linked to each other directly or via a linker or spacinggroup and/or are individually linked to D directly or via a linker orspacing group; or a pharmaceutically acceptable salt thereof. 2-13.(canceled)
 14. A method for treating a tumor or cancer in a subject,said method comprising administering to a subject having cancer anamount of prodrug or salt thereof according to claim 1 sufficient toprovide a therapeutically effective amount of drug in the vicinity ofthe tumor or cancer, said administering resulting in the treatment ofsaid tumor or cancer. 15-17. (canceled)
 18. A method for producing theprodrug according to, said method comprising the steps of: (i) obtainingthe drug; (ii) linking the drug to a capped oligopeptidic moiety,resulting m the prodrug; or, alternatively, (ii′) linking the drug to anoligopeptidic moiety followed by linking the capping group to theoligopeptidic moiety, resulting in the prodrug; and (iii) purifying theprodrug obtained in step (ii) or (ii′). 19-30. (canceled)